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Prenatal Nicotine's Effect on the Postnatal Development of Cardiorespiratory Integration in a Rodent Model

Permanent Link: http://ufdc.ufl.edu/UFE0041130/00001

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Title: Prenatal Nicotine's Effect on the Postnatal Development of Cardiorespiratory Integration in a Rodent Model Sex and State Dependence
Physical Description: 1 online resource (187 p.)
Language: english
Creator: Reynolds, Carie
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: cardiovascular, development, hypoxia, nicotine, orexin, postnatal, prenatal, rat, respiration, sleep
Veterinary Medicine -- Dissertations, Academic -- UF
Genre: Veterinary Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Epidemiological research has lead to two surprising risk factors for sudden infant death syndrome (SIDS), prenatal nicotine exposure (PNE) and gender. Because of the large risk of SIDS, PNE has become a major component of animal research to help elucidate possible underlying pathologies of SIDS. Unfortunately, little work using this animal model has attempted to elucidate any sex difference that might exist. Therefore, in the present studies we proposed to examine the potential sex differences after PNE in the maturation of cardiorespiratory integration and how the sleep/wake system might integrate it these systems. We hypothesized that in males only PNE would alter the development of cardiorespiratory and sleep/wake state integration. Our data suggest that during normal development, males and females demonstrated fundamental differeneces in ontogenetic timelines, which might account for the gender bias in SIDS incident rates. As hypothesized, PNE appeared to accelerate the integration of respiratory related regulation of the cardiovascular system indicted by an increased index of respiratory sinus arrhythmias (RSA). Additionally, we demonstrated a significantly blunted heart rate (HR) response to acute hypoxia in PNE males selectively. PNE also significantly lower HR during sleep in an age dependent manner in males alone. Using heart rate variability (HRV), PNE females altered parasympathetic indices, whereas PNE males demonstrated lower sympathetic indices. We also suggested a possible orexin mediated mechanism for these physiological changes. We found that PNE reduces orexin expression and therefore suggest a possible epigenetic pathway. Overall, PNE may cause global epigenetic changes throughout the central nervous system which changes the overall ontogenetic timing mechanisms for the integration of central processes.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Carie Reynolds.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Hayward, Linda F.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0041130:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041130/00001

Material Information

Title: Prenatal Nicotine's Effect on the Postnatal Development of Cardiorespiratory Integration in a Rodent Model Sex and State Dependence
Physical Description: 1 online resource (187 p.)
Language: english
Creator: Reynolds, Carie
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: cardiovascular, development, hypoxia, nicotine, orexin, postnatal, prenatal, rat, respiration, sleep
Veterinary Medicine -- Dissertations, Academic -- UF
Genre: Veterinary Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Epidemiological research has lead to two surprising risk factors for sudden infant death syndrome (SIDS), prenatal nicotine exposure (PNE) and gender. Because of the large risk of SIDS, PNE has become a major component of animal research to help elucidate possible underlying pathologies of SIDS. Unfortunately, little work using this animal model has attempted to elucidate any sex difference that might exist. Therefore, in the present studies we proposed to examine the potential sex differences after PNE in the maturation of cardiorespiratory integration and how the sleep/wake system might integrate it these systems. We hypothesized that in males only PNE would alter the development of cardiorespiratory and sleep/wake state integration. Our data suggest that during normal development, males and females demonstrated fundamental differeneces in ontogenetic timelines, which might account for the gender bias in SIDS incident rates. As hypothesized, PNE appeared to accelerate the integration of respiratory related regulation of the cardiovascular system indicted by an increased index of respiratory sinus arrhythmias (RSA). Additionally, we demonstrated a significantly blunted heart rate (HR) response to acute hypoxia in PNE males selectively. PNE also significantly lower HR during sleep in an age dependent manner in males alone. Using heart rate variability (HRV), PNE females altered parasympathetic indices, whereas PNE males demonstrated lower sympathetic indices. We also suggested a possible orexin mediated mechanism for these physiological changes. We found that PNE reduces orexin expression and therefore suggest a possible epigenetic pathway. Overall, PNE may cause global epigenetic changes throughout the central nervous system which changes the overall ontogenetic timing mechanisms for the integration of central processes.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Carie Reynolds.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Hayward, Linda F.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0041130:00001


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1 PRENATAL NICOTINES EFFECT ON THE POSTNATAL DEVELOPMENT OF CARDIORESPIRATORY INTERGRATION IN A RODENT MODEL: SEX AND STATE DEPEND ENCE By CARIE RENEE REYNOLDS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSIT Y OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009

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2 2009 Carie Renee Reynolds

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3 To Douglas Hayden Myers my guardian angel

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4 ACKNOWLEDGMENTS M y time as a graduate student w as not spent just learning cardioresp i ra tory physiology. Ive also learned that every good scientist, or person for that matter, stands on the shoulders of all those who have come before. I know that I could never thank all those who have got ten me to this point, but Id like to take the time of acknowledge a few. I owe the largest debt of gratitude to my mother, Robyn Hitchens She was my childhood rock (even if she couldnt grow that avocado plant!) Without my mothers love and devotion as a child, Im n ot sure w h ere Id be now. Her strength through the hardships gave me the stability to grow strong and independent myself I have dedicated my life to the achievement of the best I can be as a show of gratitude for her patience, understanding, and unconditi onal love. It is under this drive, the drive to use my talents to the best of their ability, that I became a scientist. It should not go without mention the incredible input my grandparents have had then both on my life and my mothers. It is my grandparents Jeanne and T homas Myers, who not only set everything in motion for my life, but believed in me when things got tough. I acknowle dge and most humbly thank them for bring ing me back. I also owe a thank you to Paul Hitchens. His company through the years has always provided me with light hearted yet intelligent conversation. His obvious support of my academic pursues has been a kind nod to my abilities. H e also truly deserves an award of excellence s imply because he managed to proofread some parts of the following manuscript I would also like to thank my advisor, Dr. Linda Hayward. Her patience with me through this process has been admirable. She has been the mos t incredible guide both

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5 in the laboratory and out. As I wade my way through the scientific end eavors that await me, I will always try to emulate her grace under fire. Heres to hoping I can be half the mentor she was! Also Id like to thank my committee members, Dr. Christine Baylis, Dr. Paul Davenport, Dr. Nancy Denslow, Dr. David Fuller and Dr. C h arles Wood, especially considering the length and perhaps outlandish ideas presented in the following chapters A special thanks goes to two fellow lab members, Mabelin Castellanos and Jessie Stanley. Mr. Stanley deserves a special nod because without hi m Im not sure I could have successfully developed the small animal implants. Hes superior surgerical skills only made me want to fine tune mine. Id also like to thank Lisa Fang in Dr. Woods lab who taught me the fine arts of precision pipetting and R T -PCR. T his process can at times seem lonely and arduous Therefore, I have to thank my fellow graduate student, Drs. Josyln Alhgren and Karen Porter who always understood where I was coming from and were often feeling the same. Lastly, but certainly not least, Id like to thank D r. Jeffery A. Boychuk. Without him, this process would have driven me mad long ago. He has comforted me not just through lifes physical aliments, but the emotional ones as well. H e is an amazing sounding board for both science and overall life dilemmas To come home to such a wonderfully supporting individual is a thing make in fairy tales. His constant encouragement and support was at times the only thing that got me through to the next day.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...................................................................................................... 4 LIST OF TABLES .............................................................................................................. 10 LIST OF FIGURES ............................................................................................................ 11 LIST OF ABBREVIATIONS .............................................................................................. 13 ABSTRACT ........................................................................................................................ 15 C H APT ER 1 INTRODUCTION ........................................................................................................ 1 7 Sudden Infant Death Syndrome ................................................................................. 17 Definition of Sudden Infant Death Syndrome ...................................................... 17 Role of Autonomic Regulation in SIDS ............................................................... 18 Role of Sleep -Wake State in SIDS ...................................................................... 20 Risk Factors for SIDS .......................................................................................... 22 Body position ................................................................................................. 22 Body temperature .......................................................................................... 23 Maternal smoking .......................................................................................... 24 Impact of Nicotine Exposure In Utero ........................................................................ 25 Respiratory Devel opment ........................................................................................... 27 Basal Development of Respiratory Parameters .................................................. 28 Development of the Hypoxic Ventilatory Response ........................................... 29 Development of peripheral chemoreception ................................................. 31 Development o f central chemoreception ...................................................... 32 Heart Rate Control ...................................................................................................... 33 Development of Basal HR ................................................................................... 33 Development of intrinsic HR or pacemaker activity ..................................... 34 Par asympathetic development ...................................................................... 34 Sympathetic development ............................................................................. 37 Autonomic regulation during sleep ............................................................................. 38 Physiological Function during Sleep ................................................................... 38 Development of Sl eep Patterning ........................................................................ 39 Orexin ................................................................................................................... 40 Development of orexin .................................................................................. 42 Orexin, PNE and SIDS .................................................................................. 42 Summary ..................................................................................................................... 43 Specific Aims .............................................................................................................. 44 Specific Aim 1 ....................................................................................................... 44 Specific Aim 2 ....................................................................................................... 44 Specific Aim 3 ....................................................................................................... 45

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7 Summary ..................................................................................................................... 45 2 IMPACT OF PRENATAL NICOTINE EXPOSURE ON CARDIORESPIRATORY DEVELOPMENT AND THE RESPONSE TO HYPOXIA IN CONSCIOUS RATS ... 46 Introduction ................................................................................................................. 46 Methods ...................................................................................................................... 50 Prenatal Nicotine Exposure ................................................................................. 51 Rat Pup Instrumentation ...................................................................................... 51 Data Collection ..................................................................................................... 53 Data Analysis ....................................................................................................... 54 Results ........................................................................................................................ 56 Baseline Physiology ............................................................................................. 56 RSA ...................................................................................................................... 57 Postnatal Changes in Response to Hypoxia ....................................................... 60 Discussion ................................................................................................................... 62 Sex Differences in Development of HR in CONs ................................................ 63 Sex Differences in Development of RR in CONs ................................................ 65 Sex Differences in Development of RSA in CON pups ...................................... 65 Sex Differences in the Response to Hypoxia in CONs ....................................... 66 Impact of PNE on Sex -Based Differences in C ardiorespiratory Development .. 67 Impact of PNE on Cardiorespiratory Responses to Hypoxia ............................. 70 Methodical Considerations .................................................................................. 72 Summary .............................................................................................................. 73 3 IMPACT OF PRENATAL NICOTINE EXPOSURE ON THE DEVELOPMENT OF SLEEP DEPENDENT CARDIORESPIRATORY FUNCTION ............................. 85 Introduction ................................................................................................................. 85 Methods ...................................................................................................................... 88 Rat Pup Instrumentation ...................................................................................... 89 Data Collection ..................................................................................................... 90 Data Analysis ....................................................................................................... 91 Results ........................................................................................................................ 93 Development of Sleep Patterning ........................................................................ 94 Influence of Sleep Wake State on Development of HR ..................................... 95 Influence of Sleep Wake State on Development of RR ..................................... 96 Sleeps Influence on HR and RR Variability ........................................................ 97 Male pup HRV ............................................................................................... 98 Female pup HRV ........................................................................................... 99 Male pup RRV ............................................................................................. 100 Female pup RRV ......................................................................................... 101 Discussion ................................................................................................................. 101 Sex -Based D ifferences in Sleep/Wake Development ...................................... 102 Impact of PNE on the Development of Sleep/Wake Time ................................ 103 Sex -based Differences on Developmental Changes in HR Regulation in Control Animals ............................................................................................... 104

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8 Effect of PNE on Developmental Changes in HR Control across Behavioral States .............................................................................................................. 105 Sex -based Differences the Effect of PNE on Developmental Changes in HR Control across Behavioral States ................................................................... 107 Effect of PNE on Cont rol of RR during Different Behavioral State across Developments ................................................................................................. 108 Methodical Considerations ................................................................................ 109 Summary ............................................................................................................ 110 4 IMPACT OF PRENATAL NICOTINE ON THE HYPOTHALAMIC OREXIN SYSTEM: A POTENTIAL FOR EPIGENETIC CHANGES ...................................... 123 Introduction ............................................................................................................... 123 Methods .................................................................................................................... 126 PNE E xposure .................................................................................................... 126 Tissue Collection ................................................................................................ 127 mRNA Isolation .................................................................................................. 128 RT -PCR .............................................................................................................. 129 Immunohistochemistry ....................................................................................... 130 Microscope Analysis of Immunohistrochemical Labeling ................................. 131 Physiological Measurements ............................................................................. 131 Physiological Data Collection ............................................................................ 132 Data Analysis ..................................................................................................... 132 Results ...................................................................................................................... 134 RT -PCR .............................................................................................................. 134 Orexin Cell Morphology ..................................................................................... 134 Orexin Physiology .............................................................................................. 134 Discussion ................................................................................................................. 135 Sex -Based Differences in Prepro orexin mRNA ............................................... 136 PNE Induced Changes in Prepro orexin mRNA Expression ............................ 137 PNE Effects on Orexin Cell Morphology ........................................................... 139 PNE Effects on Sleep wake Behavior and Apnea Index .................................. 140 Summary ............................................................................................................ 140 5 SUMMARIES AND CON CLUSIONS ....................................................................... 146 Summary of Findings ................................................................................................ 147 Aim #1: Impact of PNE on the Acceleration of Cardiorespiratory Function ..... 147 Aim #2: Impact of PNE on the Acceleration of Behavioral States Influence on Cardiovascular Function ............................................................................ 149 Aim #3: A Potential Orexin Mechanism ............................................................. 150 Discussion ................................................................................................................. 151 Normal Sex Differences in Central Cardiorespiratory Integration .................... 151 Effect of PNE on Cardiorespiratory Integration ................................................. 156 Effect of PNE on Sex Based Neural Plasticity .................................................. 158 PNE Increases Risk of Cardiovascular Disease ............................................... 1 59 Study Limitations ...................................................................................................... 161

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9 Surgical Stress ................................................................................................... 161 Time of Recording .............................................................................................. 161 Future Directions ...................................................................................................... 162 Autonomic Blockade .......................................................................................... 162 RSA and GABA Development in Cardiac Vagal Neurons ................................ 162 Inv estigating Multiple Risk Factors .................................................................... 163 Orexin System Development ............................................................................. 163 Conclusions .............................................................................................................. 164 LIST OF REFERENCES ................................................................................................. 166 BIOGRAPHICAL SKETCH .............................................................................................. 187

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10 LIST OF TABLES Table page 2 -1 Pup weight .............................................................................................................. 75 2 -2 Correlational values for RR vs HF location ........................................................... 81 3 -1 Average weight for all groups .............................................................................. 111

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11 LIST OF FIGURES Figure page 2 -1 Representative tracings from chronically instrumented control male rat pups illustrating the development of res piratory sinus arrhythmia (RSA) ..................... 76 2 -2 The effect of prenatal nicotine exposure (PNE) on developmental changes in baseline heart rate (HR) and respiratory rate (RR) betw een male and female offspring .................................................................................................................. 77 2 -3 Effe ct of PNE on development changes in baseline heart rate (HR) and respiratory rate (RR) relative to age and sex matched controls (CON) ............... 78 2 -4 Impact of PNE on baseline heart rate variability (HRV) components throughout development in both males and females. ........................................... 79 2 -5 The effect of PNE on the relationship between the location of the high frequency (HF) component of HRV and respiratory rate (RR) ............................. 80 2 -6 Representative tracings from one CON and one PNE male pup at postnatal day 13 during the first two minut es of an acute hypoxic exposure ....................... 82 2 -7 Effect of PNE on sex -based heart rate (HR) responses to lowering oxygen concentrations ........................................................................................................ 83 2 -8 Effect of PNE on sex -based respiratory rate (RR) response s to lowering oxygen concentrations ........................................................................................... 84 3 -1 Representative tracing of all three behavioral states at two developmental ages ( P13 and P26) in control females ............................................................... 112 3 -2 Developmental changes in the percent of total recording time spend in each behavioral state in both sexes. ............................................................................ 113 3 -3 Developmental changes in heart rate (HR) during three behavioral states ....... 114 3 -4 The effect of sleep state (NREM and REM) on heart rate changes (HR) as a function of quiet wakefulness (QW) during three developmental time point s. ... 115 3 -5 Developmental changes in respiratory rate (RR) during three behavioral states .................................................................................................................... 116 3 -6 The effect of sleep state ( NREM and REM) on respiratory rate changes (RR) as a function of quiet wakefulness (QW) during three developmental time points .................................................................................................................... 117

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12 3 -7 Representative Poincare plots of heart rate intervals ( RR) in two male pups at postnatal day 13. .............................................................................................. 118 3 -8 The effect of behavioral state on the short (SD1) and long term (SD2) heart rate variability in male pups. ................................................................................ 119 3 -9 The effect of behavioral state on the short (SD1) and long term (SD2) heart rate variability in female pups .............................................................................. 120 3 -10 The effect of behavioral state on the short (SD1) and long term (SD2) respiratory rate variability in male pups. .............................................................. 121 3 -11 The effect of behavioral state on the short (SD1) and long term (SD2) respiratory rate variability in female pups. ........................................................... 122 4 -1 Mean percent change in prepro orexin mRNA expression in the hypothalamus of 26 day old rat pups following prenatal nicotine exposure (PNE). ................................................................................................................... 142 4 -2 Representative image of the hypothalamic areas examined from a male animal. .................................................................................................................. 143 4 -3 Mean morphological measures of all male animals across all three hypothalamic areas. ............................................................................................. 144 4 -4 Physiological data from both PNE and CON male weanling rats ....................... 145 5 -1 Proposed mechanism for o rexin mediated alteration of GABA neurotransmission to cardiac vagal neurons (CVN) during normal development (CON) and after prenatal nicotine exposure (PNE). ..................... 165

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13 L IST OF ABBREVIATIONS ACh acetylcholine CO2 carbon dioxid e CON saline -treated control Ct cycle threshold CVN cardiac vagal neuron DMH dorsomedial hypothalamus ECG electrocardiogram EEG electroencephalogram f fornix GABA gamma aminobutyric acid HF high frequency component of heart rate variability analysis Hip po hippocampus HR heart rate HR SD1 short term variab ility of heart rate HR SD2 long term variabili ty of heart rate HR SD1/SD2 ratio of variability of heart rate (autonomic balance) HRV heart rate variability ICV intracerebroventricular KO knock out LC lo cus ceoruleus LF/HF ratio of the low frequency to high frequency component of heart rate variability LH lateral hypothalamus mRNA messenger ribonucleic acid mt mammiothalamic tract

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14 nAchR neuronal acetylcholine receptor nEMG nuchal electromyogram NA nucleus ambiguus NREM non-rapid eye movement NTS nucleus of the solitary tract O2 oxygen opt optic tract P postnatal day PeF perforical hypothalamus PG plethysmograph PNE prenatal nicotine exposure preB tC pre -Botzinger complex QW quiet wake REM rapid eye movement RR respiratory rate RR SD1 short term variability of respiratory rate RR SD2 long term variability of respiratory rate RRV respiratory rate varibility RSA respiratory sinus arrthymia RT -PCR real time polymerase chain reaction SIDS sudden infant death sy ndrome SWS slow wave sleep VMH ventro -medial hypothalamus W wake

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15 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy PRENATAL NICOTINES EFFECT ON THE POSTNATAL DEVELOPMENT OF CARDIORESPIRATORY INTERGRATION IN A RODENT MODEL: SEX AND STATEDEPENDENCE By Carie Renee Reynolds December 2009 Chair: Linda F Hayward Major: Veterinary Medical Sciences Epidemiological research has lead to two surprising risk factors for sudden infant death syndrome ( SIDS ), prenatal nicotine exposure (PNE) and gender. Because of the large risk of SIDS PNE has become a major component of animal research to help elucidate possible underlying path ologies of SIDS. Unfortunately, little work using this animal model has attempted to elucidate any sex difference that might exist. Therefore, in the present studies we proposed to examine the potential sex differences after PNE in the maturation of cardio respiratory integration and how the sleep /wake system might integrate it these systems We hypothesized that in males only PNE would alter the development of cardiorespiratory and sleep/wake state integration. O ur data suggest that during normal developme nt, males and females demonstrated fundamental differ enece s in ontogenetic timelines, which might account for the gender bias in SIDS incident rates. As hypothesized, PNE appeared to accelerate the integration of respiratory related regulation of the cardi ovascular system indicted by an increased index of respiratory sinus arrhythmias (RSA). Additionally, we demonstrated a significantly blunted heart rate ( HR) response to acute hypoxia in PNE males

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16 selectively. PNE also significantly lower HR during sleep i n an age dependent manner i n males alone. Using heart rate variability (HRV), PNE females altered parasympathetic indices, whereas PNE males demonstrated lower sympathetic indices. We also suggested a possible orexin mediated mechanism for these physiologi c al changes. We found that PNE reduces orexin expression and t herefore suggest a possible epigenetic pathway. Overall, PNE may cause global epigenetic changes throughout the central nervous system which change s the overall ontogenetic timing mechanisms for the integration of central processes.

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17 CHAPTER 1 INTRODUCTION Sudden Infant Death Syndrome Definition of Sudden Infant Death Syndrome Sudden infant death syndrome, or SIDS, is defined as the sudden unexplained death of an infant under a year of age. T his diagnosis of SIDS includes a complete autopsy, an investigation of the death scene, and a review of the clinical history (246) As the definition implies, SIDS infants demonstrate no major signs of distress prior to the terminal event. More startling, unfortunately, is that SIDS is considered a leading cause of infant mortality worldwide (5) and the third largest cause of infant death in the United States (146) SIDS appears to fit a distinct developmental time line. The greatest risk occurs during the first 2 4 months of age, with most deaths occurring before 6 months of age (1) At present, little is known about the causes of SIDS. The most current hypotheses suggest that infants who succumb to SIDS may be born with abnormalities in receptor expression in the brainstem and/or heart which are thought to directly contribute to unpredictable drops in heart rate, or bradycardic events which may be associated with respiratory abnormalities during or after a r espiratory challenge, such as hypoxia (121) The resulting instability of cardiorespiratory function is thought to primarily occur while the infant is sleeping and for unknown reasons are not appropriately resolved by an immediate arousal response (84) Becau se of the traumatic nature of SIDS for the surviving family members, a considerable amount of research has focused on identifying potential cardiorespiratory changes that occur in SIDS victims. Several independent researchers have

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18 documented severe bradycardias in SIDS victims prior to (77, 113, 149, 183) and during the terminal SIDS event (183). Accordingly, abnormal a utonom ic regulation of heart rate (HR), particularly within the parasympathetic branch, has been proposed as the potential cause of these bradyc ardias (108) As such it is important to note that the anatomy and influence of the autonomi c nervous system, and the parasympathetic branch specifically, is not static from birth to adulthood. For example, during the postnatal period, autonomic innervation to the heart mature s greatly In infants this maturation is indicated by an initial increa se HR during the first month, followed by a gradual drop in HR throughout the following six months (85) Role of Autonomic Regulation in SIDS HR is primarily regulated by two systems, the intrinsic pacemaker of the heart located in the sino atrial node and neural innervation to the sinoatrial node originating from the sympathetic and parasympathetic branches of the autonomic nervous system. Investigations into the influence of each branch may include an infusion of the peripheral muscarinic blocker atropine to eliminate parasympathetic inputs and/or a beta adrenergic receptor blocker atenolol to eliminate sympathetic input. Simultaneous administration of both drugs then indicates intrinsic pacemaker activity. This approach in infants has been limited, primarily due to ethical issues. Conversely, non-invasive measures, such as heart rate variability (HRV) analysis, are commonly used to examine potential changes in sympathetic and parasympathetic innervations between wakefulness or during disease conditions such as hypertension or prematurity (70, 138, 243) HRV analysis uses spectral analysis of the interval between one heart beat to the next. The resulting analysis typically yields two main frequency components of HR. The

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19 first is a high frequency peak (HF) that oscillates around the frequency of breathing and is thought to primarily reflect parasympathetic drive to the heart (17, 91) The second is a low frequency (LF) peak which is thought to primarily represent the impact of sympathetic innervation to the heart, but may also contain some index of parasympathetic drive By plotting power within each of these frequencies, HRV estimates the influence of both branches of autonomic nervous system on HR, but does not provide any index of intrinsic pacemaker activity. U sing HRV analysis, researchers have documented normal developmental increases in sp ectral power in the HF range and respiratory sinus ar rhythmias, which are attributed to increasing parasympathetic influence (190, 198) T hese changes ar e even more dramatic in preterm babies, as these infants were less developed at birth (41) In contrast, there is some evidence that victims of SIDS may have of a del ay in maturation of the parasympathetic system, possibly as a function of specific brain regions associated with cardiorespiatory integration (15) Again usi ng HRV to characterize changes in autonomic maturation, infant s who have succumbed to SIDS have demonstrated signifi cant reduction in beat -to beat variability (203) and reduced HF power compared to age matched controls (64) Hence, it has been hypothesized that dramatic alterations in the developmental time course of parasympat hetic HR control may be a strong contributing factor for the s pecific developmental window of SIDS. To date, one case study was able to correlate sleep -related changes in HRV with abnormal brainstem histology in a victim of SIDS. In this study, HRV was s hown to be below a group of control infants average only during non -rapid eye movement (NREM) or quiet sleep whereas it was higher in active or rapid eye movement (REM) sleep

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20 (119) The infant also demonstrated usually large state differences in HR After brainstem analysis, the arcuate nucleus was almost completely absent. This is important because in humans, the arcuate nucleus i s a putative central chem oreception area (61) Other studies have demonstrated more widespread abnormalities in medullary serotonin systems which included decreased receptor binding with increased cell number (120) Accordingly, th e case study presented above identified a significant reduction in serotonin binding throughout the medulla, with the largest reduction (~87%) in t he arcuate nucleus. Other studies have even demonstrated that male infant s showed greater cell loss within the arcuate nucleus which could relate to their elevated numbers of SIDS death (141) The histology of these and other SIDS infants again suggest abnormalities in chemoreception which are likely to reflect a deficit in development or neural migration. Role of SleepWake State in SIDS As n oted previously one of the single most important considerations in SIDS research is sleep state. Although SIDS infants demonstrate no major abnormality before their death, they do tend to demonstrate some subtle changes which are specific to certain sleep states (199, 200, 203) Anyone who has cared for an infant would admit that dramatic sleep pattern changes occur during the postnatal period. Adult mammals, including humans, spend approximately 30% of their time in sleep and much of that (~80%) is in a deeper stage of sleep or NREM sleep (66, 67) Although there are conflicting theories on how the specific components of the sleep/wake system develop, very young infants spen d a majority of their sleep time in active or REM sleep (71) As individuals mature, the total time spent in REM sleep begins to decrease in f avor of

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21 N REM sleep Infants at high risk for SIDS do not demonstrate normal developmental sleep patterns, but rather have smaller developmental increases in the total time spent awake and longer times spent in REM than infants with no family history or major risk factors for SIDS (34) Additionally, SIDS vi ctims have dem onstrated reduced arousal responses from s leep (86) suggesting that SIDS may occur during deep sleep state, from which it is difficult to arouse. Since arousal from sleep is regulated by intricate interconnections between different regions of the hypothalamus (195) and brainstem (211) current SIDS research has focused on identifying putative sites for brai n abnormalities. Interestingly, high risk female infants develop normal sleep patterns (33) suggesting that some of the increased risk for SIDS in male offspring may be related to sexing of the brain in utero. Even with developmental changes occurring in sleep patterning, the different stages of sleep can be identified by specific changes in cardiovascu lar and respiratory function. In particular, respiratory sinus arrhythmia (RSA), a common index of healthy cardiorespiratory interaction, is more prominent during NREM compared to REM or active wake (AW). RSA occurs during each respiratory cycle and is marked by an acceleration o f HR during inspiration and a subsequent slowing during expiration. These chan ges in HR timing are driven by multiple factors, but may be primarily associated with respiratory associated fluctuations in inhibition of parasympathetic drive to the heart v ia afferent -triggered changes in brainstem circuits. In neonates evidence of RSA is weak, but as individuals age then, regardless of sleepwake state, all measures of RSA increase (87) The lack of this physiological phenomenon highly correlates with both neonatal and adult disease and mortality (32, 149, 224) Examining SIDS infants

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22 during wakefulness often produces little significant differences. Howev er, at risk infants have demonstrated significantly higher HRs than control infant s during NREM throughout the first 6 months, while no significant HR alterations were observed during other behavioral states (85) Additionally, these at risk infants demonstrated reduced cardiac variations with respiration again during NREM sleep alone. Risk Factors for SIDS Although current evidence suggests that SI DS is the product of inappropriate cardiorespiratory integration and/or control during sleep, litt le is understood about the causes underlying the inappropriate functioning of these systems. Epidemiological studies, however, have identified several risk factors for SIDS (154) These include prenatal nicotine exposure (PNE), perinatal infec tion, sleep position and high body temperature; and all of these factors have the potential to create a cardioresp iratory response which may include a slowing of HR and wh en superimposed upon a central or peripheral abnormality or challenge, may resu lt in an unpredicted fatal outcom e. Body position As early as the 1970, body position during sleep was identified epi demiologically to play a significant role in an infants susceptibility to SI DS (50) In accordance, 50% of SIDS victims have been reported to be found with their face covered by bedding (116, 233) and in most cases, the infant is found with their face straight or near straight down, a position only po ssible if placed in the pr one position (117, 159) Several theories have been proposed as to the physiology of the dramatic impact of prone position on SIDS death, which range from simple suffocation from bedding to changes in arousal thresholds and vestibular influences while lying prone. Indeed there i s evidence that mean arterial pressure drops significantly during a head tilt challenge in the prone

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23 position compared to pre-tilt pressures in healthy infants, specifically at 2-3 months of age (251) Although these abnormal responses are prominent in all sleep states, NREM appears to be the most compromised. During this same developmental time frame (2-3 months), normal infants demonstrate elevated HRs in the prone position c ompared to supine during sleep (250) Following the identification of prone sleeping as a significant risk factor for SIDS, in 1992 a Back to Sleep campaign was undertaken, which advised new parents to place their newborns in the supine position during sleep. Not so surprisingly then, since 1992 the incid ence of SIDS has declined by approximately 50% (154, 156) Body temperature Of equal importance to the discussion of SIDS is the potential role of high body temperature on an increased risk f or SIDS. SIDS is one of the few diseases that demonstrates a seasonal trend as there is an increase in sudden infant deaths during the winter months when infants are bundled heavier (204) Since then, SIDS victims are often found to have higher body temperatures than control infants (223) be wrapped heavier, and in warmer houses than controls (62) Interestingly, heavy wrapping and a viral infection together create a very high risk for SIDS (74) ; suggesting again that inappropriate body temperature regulation may decrease an infants ability to respon d to hypoxia. Furthermore, at 3 months of age, there is central reorganization of brain systems involved in thermoregulation which make infants particularly vulnerable to hyperthermia (9) Few studies have looked at temperature related cardiovascular changes in infants. However, apnea frequency (36) and periodic breathing during REM sleep (20) has been reported to increase during warm exposures.

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24 Maternal smoking The single largest risk factor, however, is prenatal exposure to cigarette smoke. Maternal smoking is correlated with a four -fold increase in an infants risk (155, 157) Even more startling is the observation that despite knowledge that cigarette smoke is harmful to not only mothers, but their unborn infants, women are continuing to smoke during their pregnancies. In fact in a recent National Institute of Drug Abuse survey, 23% of women of childbearing age had smoked a cigarette within a month of being surveyed and 16.4% of pregnant women reported that they smoked during their pregn ancy (227) Even with unclear causation, current evidence, to b e reviewed below, suggests that nicotine, a major constituent of cigarettes is the primary culprit mediating the increased risk for infant mortality. Accordingly, nicotine has been sho wn in the serum of some SIDS victims (151) In addition to increased risk of SIDS, nicotine has been associated with low birth weight (44) and other developmental abnormalities, including attention defect disorders (150) I nterestingly, there is now an established link between low birth weight and cardiovascular disease later in life (13) suggesting that some of the impact of PNE on autonomic regulation may persist beyond the first six months of life. In view of the strong link between PNE and SIDS, an animal model has been developed to identify putative neural factors which may contribute to SIDS. This mo del was developed by Slotkin and colleagues (168) and has b een used over the last 20 years by many investigators. This model of PNE has proved to be an invaluable tool for understanding SIDS related changes in receptor function and impact. The following data presented below will be a review of what has been lear ned abou t nicotine and its ability to alter both cardiorespiratory function and integration.

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25 Impact of Nicotine Exposure In Utero Nicotine, itself, is an exogenous ligand for the nicotinic acetylcho line receptors (nAChR). The nAChR is a ligand -gated ionotr ophic channel which consists of five subunits. When thes e receptors have bound two molecules of acetylcholine (ACh), the channels open to allow the passage of cations. This influx depolarizes the membrane, opens voltage-gated channels and allows the additi onal influx of calcium. Although neuronal nAChR de sensitize more rapidly compared to their counterparts at the neuromuscular junction, the exact rate of desensitization depends on the type of subunits present in the receptor. T he ac tivation of nAChR by ni cotine is a longer lasting response because, unlike endogenous acetylcholine (ACh), neurons do not contain an enzyme that readily degrades nicotine and therefore its overall metabolism is slow (173) nAChR are widespread throughout both the adult and fetal nervous system, including areas critical to sleep, autonomic and respiratory function (37) Nicotine, the major constituent of cigarettes and a known neuroteratogen, readily crosses the maternal placental bar rier. Using rodents, research ers identified that nicotine i s first able to bind to medullary nicotinic receptors around gestational day 12 to 14 (229) Though th e binding ability of nicotine decreased slightly during late gestati on (161) the brainstem and midbrain show a marked increase in binding capacity within the first 3 weeks after birth in the rodent (126) It was during this postnatal time point that PNE pups demonstrated up-regulated nicotine binding in the brainstem and midbrain at least until postnatal day (P)15 (214) The up -regulated binding may be a compensatory mechanism for receptor desensitization. However these effec ts are most intriguing because the nicotine infusion was ended at birth. Therefore, the effect of PNE on nAChR binding was sustained even after the drug was removed. These changes in

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26 receptor binding have been shown to have a functional consequence. Within the brainstem, there was an altered intracellular response to promote inhibitory signals and attenuate stimulatory ones by actions on adenylyl cyclase (215) The effect of PNE has been implicated in more than just the development and responsivenes s of nAChR PNE also appears to alter the function of several of the monoaminergic systems. For example, norepinep hrines utilization rate in the hindbrain and midbrain has been reported to be increased during early postnatal development in PNE rodent offspring compared to contr ols, while the utilization rate of this neurotransmitter appears to be become deficient by w eaning (168, 169) Additionally, the hindbrain and midbrain showed an increased capacity for serotonin binding following PNE (248) N icotine also has vasoconstrictor actions. Nicotines vasoconstrictor abilities are probably two-fold. First, nicotine appears to mediate indirect actions on vascular tissue via stimulation of sympathetic postganglionic neurons a nd subsequently cause s the release of catecholamine (236) Second, nicotines direct actions of vasculature are less clear, but previous studies have demonstrated that nicotine can inhibit voltagedependent K+ channels in rodent tail arteries (231) It was no surprise then that maternal injections result in transient maternal vasoconstriction which triggers some level of fetal hypoxia (218) In fact, many researchers have argued that the primary eff ect of PNE in the model established b y Slotkin and colleagues may simply be a result of a hypoxic env ironment created by nicotines vasoconstrictor actions on the umbilical artery. Moreover, prenatal intermittent hypoxia has been shown to reduce the autoresuscitation response in postnatal pups (79) However, the use of slower constant

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27 nicotine release, by way of osmotic mini pumps eliminated the fetal hypoxia associated with maternal nicotine administration (218) Although the question of a possible direct action of nicotine on brain chemistry and development remains, several neurotransmitters are known to act as neurotrophic factors during development. Within the brain, alterations in the levels of these trophic facto rs have the possibility to affect the fetus not only during the first trimester, but also throughout the pre and postnatal periods (213) Not surprisin g then, during early neurulation, maternal nicotine infusion has been shown to increase the number of pyknotic cells particularly within the areas destined to become the hindbrain, even in concentrations that did not show evidence of any other markers for developmental delays (188) Additionally, investigators found that areas of increased cell death showed increased mitotic cell numbers especially within the hindbrain regions. This sugges ts that PNE may also tri gger premature differentiation of cells during neurulation and the increased mi totic index could be a compensatory mechanism for the lowered cell counts. These effects on brain development have been shown to last longer than origina lly thought. Alterations in both the serotonin and acetylcholine systems follow ing PNE can still be documented during adolescence (217, 220, 248) and adulthood (217) in the rodent brain. Respiratory Development The respiratory system functions in concert with the cardiovascular system to maintain homeostasis both during basal, or resting conditions, and during stress Neural control of these systems is not fully developed at birth in either rodents or infants; and since SIDS is possibly attributable to an inappropriate develop ment of the integration of these two systems, it is important to understand normal development of both systems.

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28 Since the pharmacological and epidemiological data suggests that nicotine (at least exposu re in utero) alters overall neural maturation and function, it is also important to understand how P NE can affect that development. Basal Development of Respiratory Parameters During the early postnatal period, rodent pups undergo a large amount of developmental changes in basal respiratory parameters. Though the exact time course of pe ak respiratory rat e (RR) development is some what conflicting (96, 13 4) most studies agree that it occurs somewhere during the second w eek of life in rodents and declines through the third. Respiratory cycle timing is driven by a central pattern generator located in the brainstem which integrates sensory inputs with descending commands to create an appropriate RR (189) It is possible that disagreements in the exact timing of RR development may be a function of the behavioral state during the recording period, since i t is known that RR is highly affected by arousal state in both adults and infants (38, 130, 166) Additionally, increases in absolute tidal volume throughout the postnatal pe riod have been documented. Since minute ventilation is a function of both tidal volume and RR, developmental or strain related differences in tidal volume could markedly influence RR. Absolute tidal volume does increases throughout the postnatal period, but since tidal volume is related to body weight when this increase is corrected for the developmental increase in body weight, tidal volume actually decreases with age (134) Associated with changes in overall function, the postnatal period also demonstrated dramatic changes within the lungs, specifically the alveoli (29) and the neurotransmitters in brain regions critically important in the production of rhythmic ventilation, including both the

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29 p re -Btzinger complex (preBtC) and the dorsal medull a or nucleus of the solitary tract (NTS) (135-137) Currently, there are conflicting repo rts on PNEs effect on normal, or eupenic, minute ventilation throughout development. Some studies have demonstrated increases (143) decreas es (212, 222) or n o changes (10, 96) These studies also disagree on PNEs effect on RR and tidal volume Al though most agree that PNE can a ffect the se variables in some way and more importantly at specific time points (96, 143, 212) a discussion of possible reasons for these discrepancies follows in the next section. Development of the Hypoxic Ventilatory Response In the 1990 s, the first real link between the role of PNE in SIDS was identified when it was document ed that PNE increased rodent neonatal mortality in response to a hypoxic challenge (216) Hypoxia, itself, is monitored through both peripheral and central chemoreceptors, which are sensitive to low pH and O2. Peripheral chemoreceptors are located at the level of the bifurcation of the common carotid artery in the carotid body and are innervated by the petrosal ganglion (within the glossopharyneal nerve). Carotid sinus nerve afferents terminate in the dorsal medulla, in the caudal NTS (72) Alternatively, the exact locations and contributions of central chemoreceptors to the hypoxic ventilatory response (HVR) are up for debate. Yet, it is clear that, in general, the central chemoreceptors are restricted to the lower brain and include the ventral surface of the medullar, the NTS, the locus coerulus, the ventral respiratory group and the medullary raph (166) These receptors are also activated by changes in pH and O2, yet what these receptors are sensing is also up for debate. Although some believe these receptors are sensiti ve to oxygen levels in arterial blood based mainly on their proximity to blood vessels (26) there is strong evidence that

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30 these receptors are instead monitoring the interstitial fluid in the brain (164) Because these interstitial fluids are poorly buffered, small influxes of CO2 cause large changes in pH. In an adult mammal, the HVR occurs in two phases. The initial phase (pHVR) includes an increase in ventilation, which is the res ult of peripheral carotid body receptors stimulation. The second phas e is a depression of the ventilation (HVD) which is thought to be a result of pH changes (caused by the drop in CO2) either in the systemic blood or in the brain s interstitial fluid as a result of the hyperventilatory response during pHVR. Immediately after birth in the rodent, the pHVR is virtually absent (80) However, the pHVR increases with increasing postnatal age, reaching a peak response at ~P10 while the HVD response dec reases. Unfortunately, the data on the effect of PNE on the HVR are as conflicting as the impact of PNE on eupenic ventilation. Some studies have demonstrated that PNE decreases a neonatal rats ability to change tidal volume (212) in response to hypoxia, while others demonstrate increased t idal volume responses (143) or no change (10) Although these studie s disagree on the impact of PNE of respir atory timing changes in response to hypoxia, mos t studies do agree that P NE alters RR responses under hypoxia at some level. The potential cause of these conflicts may be related to differences in temperature, nicotine dose administration, and/or age group i ng between studies. For example, temperature reported for all studies evaluated above were from 29 32 C in animals aged P221. Therefore, these studies covered a large rang e of thermoneutral temperatures for developing pups (67) T emperature, s pecifi cally hyperthermia, has recently been shown to alter the hypoxic response in pups exposed

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31 to cigarette smoke compared to cont rol (178) Altern atively, the method used for nicotin e administration has also been slightly different between each study. One study in particular used an implanted nicotine pellet instead of the commonly used osmotic mini pump (143) Additionally, only one study identified what mate rnal weight was used to determine nicotine dosage (212) A pregnant rat dam gains more than 53% of her pre pregnancy body weight during gestation. Sinc e nicotine dose is based on maternal body weight, i t is important to know the weight that was used to calculate dosage as it can change the relative dose delivered to the pups in utero. Finally, most studies have differed substantially between the age groups examined and how age ranges were grouped. Since respiratory patterning can change dramatically in a one to two day postnatal period up to the time of weaning (134) it is important to maintain small age ranges within groups. All these factors must be considered when comparing result s between studies. Development of p eripheral c hemoreception In the neonatal rat, both the carotid body and central chemoreceptors are immature. The developmental changes associated with the HVR correlate with the two distinct phases of carotid body development (102) The first stage occurs immediately after birth and is referred to as the silencing phase. During this stage, the peripheral chemoreceptors are virtually unresponsive to hypoxia. It is thought this is due in large part to a resetting of the hyp oxic threshold requir ed in the transition from fetus to infant (72) The next phase is a gradual increase in responsiveness. With increasing age, the carotid bodies afferents demonstrate increasing sensory discharge with similar levels of oxygen tension (30, 144)

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32 Peripheral chemoreceptor s have nAChRs (4) Thus, it is not surprising that PNE a ffects carotid body chemoreceptor development. In animals exposed to PNE, tyrosine hydroxylase (TH ) mRNA expression in both the carotid body and the petrosal gangli on have been reported to be higher during PND 0-3 compared to controls (73) Additionally, dopamine -hydroxylase expression has been shown to be increased at P15 following PNE exposure, but only within the carotid body. In normal development, TH activity d ecreases within the first week and maintains that level until at least P21 (89) In PNE, however, this decrease doesnt occur until approximately P11 (143) In normal development, then the decrease in TH activity indicates a decrease in dopamine content and an increasing responsiveness to hypoxia through the postnatal period since dopamine within the carotid body attenuates the hypoxic chemosensi ti vity. The delayed blunting of TH activity within the first week after PNE is associated with a blunti ng of chemosensation (because dopami ne levels are too high). Therefore, PNE animals would demonstrate a blunted hypoxic response when compared to controls, at least during early development. Development of c entral c hemoreception Peripheral chemoreceptors are not the only site for hypoxia detection or response regulation, nor are they the only site for potential PNE modulation of the HVR. In many brainstem sites, including the NTS, ventral lateral medulla and locus coerulus, populatio ns of catecholaminergic cells known to respond to hypoxia w ith increased c Fos expression ( a marker of neuronal activation) are present at birth but activated cell numbers do not peak until P 1015 (18, 244) Central TH activity is also reduced between P7 to P11 in several catecholaminergic brainstem sites responsible for central chemoreception, including the locus coerulus (143) Additionally, areas known to affect

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33 cardiovascular func tion (though not directly involved in chemosens a tion, but probably help coordinate the cardiovascular response) such as the pontine parabrachial nucleus and the dorso medial hypothalamus (DMH) did not show evidence of neuronal activation during hypoxia at birth, but d id s tart showing indicators of activation at P10 15 (18) Although many of these brain regions are not directly involved in central chemoreception, these data suggest that central integration of the hypoxic response is developing throughout the postnatal period. Additionally, after an acute exposure to hypoxia, PNE pups did not demonstrate an increase in TH mRNA in the locus coerulus, which is seen in control animals (245) In mice PNE has been shown to attenuate the fictive breathing response to acidification of the solution surrounding the brainstem (56) Additionally, in this study cholinergic contributions to this sensation were found to have been switched from primarily m uscarinic to nicotinic in PNE mice. Thus, the general consensus from animal work is that PNE decreases an animals ability to respond to a chemosensitive challenge by alterations in both peripheral and central chemoreceptors. Heart Rate Control Developmen t of Basal HR Rodent pups demonstrate increasing resting HRs throughout the postnatal period and similar to humans, during this same time period rodents develop increasing parasympathetic drive (114) Also like infants, as rodent pups mature, there is an increase in HRV power, specifically in the HF or parasympathetic range, and an increase in the strength of the cardiorespiratory interaction, or RSA (114) As with respiratory de velopment, the effect of PNE on HR is also conflicting in the literature. In some studi es, PNE has been reported to induce a reduct ion in HR (59) while in other studies HR was reported to increase (83) or showed no change (219)

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34 These differences could be attributable to the sp ecies used, sex, and/or behavioral state. However, none of the studies mentioned have examined HR development in PNE animals beyond P10, which is a huge experimental flaw since parasympathetic drive does not appear to play a significant role in HR regulati on until P15. Furthermore, P15 is probably the developmental point in the rodent that is more closely related to the critical development time window for SIDS infants, between 2 -12 months of age (186) These changes in basal HR bot h in normal development and with PNE are a product of changes in both HR cont rol systems mentioned previously. The f ollowing discussion will briefly review how each component of these two systems are changing throughout development with a primary focus on how PNE alters that development. Development of i ntrinsic HR or p acemaker a ct ivity The hearts intrinsic rate is determined by the spontaneous depolarization of the fastest cardiac pacemaker cells which are located in the sino artial node within the posterior wall of the right atrium. Without neur al innervations, this rate is the r esting HR. In rodents, intrinsic HR increases throughout the postnatal period (237) The increase in intrinsic HR is balanced by developmental changes in autonomic drive, such that between P13 and P21 resting HR does not change dramatically. Unfortunately, to date no work has been done on PNEs potential impact on postnatal changes in intrins ic HR. Parasympathetic d evelopment The parasympathetic system influences the heart by way of a postganglionic connection in the vagus nerve. The vagal input to the sino atrial node of the heart is generated from a region of the ventrolateral medulla known as the nucleus ambiguous (NA) (240) By releasing (or withholding) the release of ACh, this branch of the autonomic nervous system slows the rate at which the heart beats (or sp eeds it up). In

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35 the adult, the parasympathet ic nervous system is the dominant determinant of basal HR. T onic parasympathetic inp ut to baseline HR does not occur in the rat until P 15 (114) This is evidenced by a lack of change in HR when a peripheral muscarinic receptor blocker is given. It is only after P15, that systematic administration o f a cholinergic antagonist, such as atropine, begins to increase basal HR (242) Comple te adult like maturation of parasympathetic or vagal tone is seen only as early as P30 in rodents (162) As mentioned above, the development of RSA is a good indicator of the maturity of the parasympathetic system. RSA facilitates appropriate ventilation and perfusion ratios throughout the entire respiratory cycle (24) Though RSA is related to pulmonary stretch receptor activity and the mechanical effect of respiration on both venous return and stretch of cardiac pacemakers, the parasympathetic nervous system and central respiratory pattern generator play a well documented role in its generation as well (6, 171, 179) Examinations into the neural circuits involved i n the generation of RSA have demonstrated that preganglionic parasympathetic fibers from the NA that innervate in the heart are typically silent during inspiration, but fire more rapidly during post inspiratory events (123) This pattern can also be observed when the cardiac vagal neurons within the NA are recorded from directly (75) In recent years, the Mendelowitz lab has utilized the neonatal brain in vitro slice to investigate the cellular mechanism underlying RSA in both control animal s and PNE offspring. In control pups, during inspiratory -bursting recorded from hypoglossal rootlets, cardiac vagal neurons have been shown to demonstrate no inspiratory -relat ed excitation. However, the frequencies

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36 of both inhibitory GABAergic and glyinergic synaptic events increased significantly, suggesting the neural circuitry is sufficient for generating RSA, and an increase in HR via a decrease in parasympathetic drive, is present in the brainstem (171) Fur thermore, focal application of a specific nicotinic receptor antagonist has been shown to abolish the respiratory related increase in GABAergic events (171) Th e sensitivity of cardiac vagal neurons to nicotine application has been suggested as playing a role in the elevated risk of SIDS with PNE (98) It follows the n that PNE has been shown to inhibit normal inspiratory r elated GABAergic neurotransmission during normoxia (98) However, cardiorespirator y integration occurs not just under basal conditions, which is critical to remember when studying SIDS, given that SIDS victims appear relatively norm al in the absence of challenge. Prev ious examinations of SIDS infants have demonstrated a failed ability t o autoresuscitate to a hypox ic stimulus (113, 149, 183) Furthermore, during hypoxic challenges, PNE animals (both rodent and lamb) have demonstrated a reduced HR response (83, 21 9) Again using in vitro studies, the activity of cardiac vagal neurons under hypoxia has been investigated. In control preparations exposed to hypoxia/hypercapnia, cardiac vagal neurons initially demonstrated synaptic events similar to those seen during basal conditions such as increases in inhibitory neurotransmission during inspiration. However, the response is bi phasic and eventually, the response switches and inhibitory neurotransmission decreases, while exc itatory transmission remains unchanged (171) The two phases of intracellular c hanges in cardiac vagal neurons are proposed as the cellular mechanism for the two phases of the HR response t o hypoxia observed in vivo

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37 During an acute hypoxic exposure, cardiac vagal neurons demonstrate a biphasic respon se. Initially, the drop in oxygen concentration causes a tachycardia (90, 145, 219) If the hypoxic stimulus is maintained, the second phase involves a graded bradycardia. In the brainstem this might correspond to an initial increase, followed by a decrease in GABAergic input to cardiac vagal motoneurons. D uring hypoxia/hypercapni a, in vitro, PNE triggers an abnormal recruitment of an excitat ory glutaminergic inp ut and decreased inhibitory input. Additionally, during recovery from hypoxia, PNE abolishes a ser otonergic pathway and instead recruits a glutamatergic pathway (108) T he inhibition of inhibitory neurotransmitter and the recr uitment of excitatory pathways are the proposed mechanism s for the blunted HR response seen in the hypoxic response of young PNE animals. Sympathetic development In addition to changes in parasympathetic drive and i ntrinsic pacemaker activi ty, basal HR during development is also influenced by sympathetic innervation or circulating catecholaminergic levels. The cell bodies of pre-ganglionic sympathetic neurons are located within the intermediolateral cell column of t he upper spinal cord. These pre ganglionic neurons release ACh and synapse with the post -ganglionic sympathetic neurons that innervate the sinoartial node, atrium and ventricle. The post ganglionic sympathetic nerve terminals release norepinephrine which accelerates the rate of depolarization of the pacemaker cells and antagonizes vagal nerve drive input, thereby inducing an increase in HR. Unlike the parasympathetic system, the sympathetic system exerts significant control over HR during the postnatal p e riod, which is evidenced by a significant drop in HR caused by adrenergic receptor blockade during the first postnatal week in rat pups

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38 (3, 237, 238) Although the sympathetic system influences HR during very early postnatal development, the cardiac innervations are not fully developed until the second week (8) As a possible compensation to immature sympathetic innervations, cardiac adrenergic receptors actually demonstrate increased sensitivity at birth and reuptake systems are minimally active (12) As sympathetic innervation matures, the cardiac receptors decrease sensitivity. Since similar compensatory mechanisms have not been found in the parasympathetic system, HR is predominately controlled by sympathetic activity at very early postn atal days. Autonomic regulation during sl eep Sleep is often considered an important physiological function for a wide range of behaviors, including overall health and memory. Four main behavioral stages exist, active wakefulness (AW), quiet wakefulness (QW ), NREM and REM. Though the complexities of the sleepwake networks and their interplay between each state will not be discussed here, its important to remember how proper sleep regulation can contribute to the health of an individual. Very specific chang es occur in physiological parameters as an individual, regardless of s pecies, transitions between wakefulness and sleep. Physiological Function during Sleep When measured for 24 hrs, both mean arterial pressure and HR can show circadian rhythms (207) In hu mans, HR dips during sleep compared to wakefulness, meaning as an individual goes from wake to sleep, HR values drop by approximately 10%. Clinically this is important because an attenuated capacity to produce heart rate dipp ing during sleep has been shown to be an independe nt predictor of mortality,

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39 particularly in hypertensive patients (16) A dditionally, baroreflex sensitivity changes with behavioral state (256) As with HR, respiratory patterns have also been found to vary according to a circadian rhythm (209) and with sleep/wake state (226) NREM also demonstrates a marked decrease in CO2 sensitivity, suggesting that chemoreceptor function (specifically CO2, but maybe O2 as well) is altered simple as a function of sleep (28) Investigating these broad effects has revealed that many central respiratory control regions ha ve state dependent functionality. For example, unilateral lesioning of substance P containing neurons in the preBtC significantly increases the incidences of respiratory disturbances, mainly central apneas and hypop neas, but only during REM sleep (148) Furthermore, selective lesioning of the brainstem catecholaminergic neurons ha s been reported to slow breathing frequencies to room air and CO 2 during both wakefulness and NREM sleep, but not during REM (129) Interestingly, this study examined breathto breath variability and found that variability was increased only during REM sleep, suggesting again that breathing regulation is complex and statedependent. Development of S leep Patterning Much like autonomic function changes over the course of an individuals lifetime, so does on es pattern of sleep. Rodent pups, however, do not demonstrate all adult like sleep charact eristics nor are circadian related sleep patterns present until P16 (67, 68) approximately the same developmental time point that parasympathetic regulation of HR becomes noticeable. In particular, after P16 there i s a general incr ease in the length of time spent in w akefulness and a decrease in the time spent in REM with increasing age (68) Even specific sleep regulatory centers of the brain, like the

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40 suprac hiasmatic nucleus, do not demonstrate sleep / wake cycles until the 3rd postnatal week in rodents (127) Identifying chang es in the development of sleep/ wake patterns may be important to gaining greater insights into mechanisms underlying SIDS. Indeed, infants at high risk for SIDS have demonstr ated impaired ability to arouse from sleep compared to normal i nfants (86) possibly suggesting a change in NREM sleep development. Th e effect of PNE on sleepwake behavior in animal models has been explored in few studies. In general, PNE accelerates the development of circadian rhythms (68) PNE pu p spend significantly longer time awake in general compared to controls. They also demons trate significant differences in sleep/wake times according to dark/light cycles and develop these patterns approximately three days earlier than control pups. More sp ecifically, following exposure to maternal cigarette smoke, neurons in the pedunculopontine nucleus (PPN) have been shown to be hyper excitable at increasing postnatal ages compared to control pups (76) The PPN is part of the r eticular activating system which has been implicated in the control of arousal, wakefulness and REM sl eep. Orexin One interesting neuropeptide with function in sleep/wake cycles, arousal and autonomic function is orexin. Orexin (also known as hypocretin) was first discovered in 1998 as an integral player in the control of feeding behavior (40, 193) I ts role in the promotion of wakefulness was firs t demonstrated when a mutation in an orexin receptor was linked to canine narcolepsy (133) Narcolepsy manifests as chronic sleepiness with marked disorganiz ation of sleep/wake behavior which usually begins in humans around adolescence (196) Orexin acts as a promoter of wakeful ness and sleep suppressor and

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41 orexinergic neurons receive projections from the main circadian rhythm generator, the suprachaismatic nucleus. It is thought that these projections are important in maintaining sleep related circadian rhythm because narcolepti c individuals de monstrate normal circadian rhythm in other physiological systems outside of sleep/wake behavior. Therefore, narcoleptic sleep disturbances are pr obably not related to a dysfunction of the suprachaismatic nucleus. In addition to sleep wake regulation, orexin appears to have an additional function as an autonomic modulator. Anatomically orexin neurons are located in the lateral and the dorsomedial hypothalamus (LH and DMH respectively) with projections to importa nt cardiovascular centers in other regions of the hypothalamus and brainstem (180) Interesti ngly, the DMH (and therefore possibly the orexin neuron within it) plays a critical role in the cardiovascular response to stress. It has been shown that intracerebral ventricu lar (ICV) administration of orexin increases mean arterial pressure, and elicits a tachycardia in the conscious rat (210) Subsequently, a dministration of orexin followed by intravenous (IV) injection of an alpha(1) adrenergic receptor antagonist or a beta adren ergi c receptor antagonist attenuated the increase in mean arterial pressure and HR (7) Though done in anesthetized mi ce, central administrat ion (intracerebroventricular, ICV) injection of orexin A has been shown to increase not just HR and blood pressure, but respiration as well (254) Accordingly, when exposed to hypercapnia, orexin knock out mice demonstrate a blunted ability to increase minute ventilation during wakefulness, but not sleep (163) Alternatively, orexin knock out mice do not appear to demonstrate any change in the response to a hypoxic challenge. However, this study did not examine the car diovascular response components in these

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42 mice to either stim ulus. Since orexin typically causes a tachycardia, it is possible these animals also demonstrated a blunted HR response to either hypoxia or hypercapnia, but this remains to be determined. If so, alterations in the orexin system or development of the orexin system by PNE could also contribute to the blunted HR response to hypoxia typically seen after PNE. Development of o rexin Orexin neuronal development occurs on a similar time course as general sleep development. At parturition in the rat, orexin levels in the hypothalamus are very low (225) At approximately, P13 orexinergic neuron levels begin to increase until P 26. At this point no significant differences exist between the adolescent and adult, though orexin levels do appear to drop slightly with incr easing age (225) Postna tal changes in orexin levels also coincide with changes in overall sleeping behavior. Interestingly, orexin knock out (KO) mice appear to lag behind in circadian rhythmicity, by demonstrating no change in sleep or wake bout duration by P21 (22) Additionally, the KO mice demonstrated no significant differences in sleep from P4 to 12, suggesting that orexin dependent changes in sleep/wake behavior follow the development of these neurons in the LH and DMH. Orexin, PNE and SIDS Although the role of the orexin sy stem in both sleep/wake behavior regulation and cardiorespiratory function in response to physiological challenges suggests that this peptidergic system might play an important role in SIDS, to our knowledge the role of orexin in SIDS has not yet been eval uated. Furthermore, to our knowledge the effect of PNE on the orexin system and how that might also play a role in increasing the risk of SIDS has also not been previously evaluated. However, PNE has been demonstrated to

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43 in nAchR in the hypothalamus from P12 and 14 which is a developmental time period important for the maturation of the orexin system (68) Moreover, the in utero and is associated with marked calcium permeability which is implied in the role of this subunit association with synaptic plasticity (49) Furthermore, acute administration of nicotine in adults rats increases c -fos expression in orexin containing neurons of the LH and that response i s attenuated by application of a nicotine antagonist (176) Finally, chronic nicotine treatment in adults increases both preproorexin levels (specifically in DMH) and its receptors, orexin 1R and 2R, mRNA levels (110) but decreases the affinity of orexin -1R binding (109) Therefore, these two findings suggest that nAChR may be important in the development of orexin neurons and their function and that the time course of this development may be altered by PNE. Summary In summary, SIDS is a tragic developmental disorder t han affect s not only the infant, but the family left behind. Current research suggests SIDS may be a result of an abnormal cardiorespiratory re sp onse to physiological challenges during sleep, such as hypoxia. PNE appears to mimic the characteristics for this disease and therefore provides an excellent animal model that allows researchers to investigate potential causes and treatments for SIDS. However, much research is needed to fully characterize the abnormalities, particularly those related to cardiovascular development as a function of sleep/wake state. To date, research has identified that PNE may cause changes in respiratory developmental pa tterns, a blunting of cardiovascular responses to hypoxia (83) and alterat ions in cardiac vagal neurotransmission both duri ng both normoxia and hypoxia/hypercapia (97) Sleep appears critical to the understanding of

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44 PNEs effect because both sleep/wake patterning and state-dependent cardiac function are altered by PNE in rodents (68, 83) Althou gh, there is evidence for wide-spread brainstem receptor abnormalities with SIDS and PNE, few studies have examined hypothalamic sleep/wake regulatory neuropeptides and their potential r ole in this tragic event. E ven more important however is that few studies have examined PNEs effect on rodent development outside of the first week of life which is the time more consistent with postnatal human developm ent and the critical time window for SIDS (186) Base d on the information provided above, th e group of studies listed below were undertaken to evaluate for the first time the impact of PNE on state dependent changes in cardiorespiratory function. Specific Aims Specific Aim 1 Determine if PNE affects cardiorespiratory integration and response to hypoxia during early postnatal development in the rodent. Hypothesis : PNE will induce a sex dependent accele ration of cardiorespiratory integration and blunted HR responses to hypoxia at P13 and P16 but by P26 (post weaning) PNEs developmental abnormalities will be r esolved. Specific Aim 2 Determine if PNE affect s the development of state dependent changes in c ardio vascular and respiratory system function throughout early postnatal development in rodents. Hypothesis : PNE will induce a sex -dependent acceleration of developmental changes in HR associated with sleep/ wake state at P13 and P16 but will not alter deve lopmental changes in RR. By P26, PNE induced developmental abnormalities will be resolved.

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45 Specific Aim 3 De termine if PNE can affect orexin cell development in the hypothalamus, specifically neuronal morph ology and mRNA expression. Hypothesis : Male PNE animals will demonstrate lower orexin mRNA, decreased cell size but increased cell number (particularly within the DMH) compared to females and control counterparts. Summary The overall goal of the present body of work was to characterize sex based differe nces in the effect of PNE on the development of cardiovascular and respiratory system s beyond the first week of life in a rodent model of SIDS. Since males m ore frequently succumb to SIDS, we hypothesized that sex -based differences in cardiorespiratory int egration must exist during early postnat al development. F ew previous studies in rodents have specifically evaluated sex based differences, particularly in regards to autonomic development. Previous studies have demonstrated that PNE significantly blunts c ardiovascular function during the first days of postnatal development. We hypothesized that male PNE pups would have significantly blunted HR responses to hypoxia as a function of accelerated cardiorespiratory integration, which female PNE pups would not have compared to their counterparts. Finally, since SIDS infants often present with cardiorespiratory abnormalities during specific sleep states, we further hypothesized that again only males would demonstrate an accelerated state dependent drop in HR duri ng early postnatal development. As a potential mechanism of these changes, we examined PNEs effect of the cellular morphology of hypothalamic orexin containing neurons. Since t his neuropeptide is important in sleep/wake regulation, we hypothesized that i t would be altered after PNE.

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46 CHAPTER 2 IMPACT OF PRENATAL N ICOTINE EXPOSURE ON CARDIORESPIRATORY DEVELOPMENT AND THE RESPONSE TO HYPOXIA IN CONSCIOUS RATS I ntroduction In a recent National Institute on Drug Abuse survey, 23% of women of childbearing age had smoked a cigarette within a month of being surveyed and 16.4% of pregnant women reported that they continued smoking during their pregnancy (227) These statistics are startling, considering that epidemiological studies have established that prenatal exposure to cigarette smoke increases an infants risk of SIDS by four fold (157) Empirical evidence suggests that the nicotine in cigarette smoke is the primary culprit mediating the increased risk of SIDS (216) Accordingly, nicotine has been identif ied in the serum of some SIDS victims (151) Although SIDS incidences have declined recently, it is still considered the leading cause of infant mortality in developing countries (146, 155) Another striking risk factor for SIDS is gender. Male infants are more likely to succumb than female counterparts (158) It may be important then to consider how these two factors (gender and nicotine exposure) are acting both independently and in tandem to increase the risk of SIDS. The nicotine contained in cigarettes is a known neuroteratogen that readil y crosses the maternal placental barrier (139, 140, 2 30) At doses equivalent to those seen in moderate smokers, prenatal nicotine exposure selectively alters early embryonic neural differentiation versus somatic differentiation, resulting in increased mitotic versus meiotic events (188) Additionally, nicotine has been s hown to alter later development of larger organ systems, including overall lung function (181, 194) cardiac receptor sensitivity (27, 167) and alteration of several brain neurotransmitter systems, such as acetylcholine, epinephrine, serotonin, and dopamine (169, 214, 248) Many of

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47 these neurotransmitter systems are critically important in bot h cardiovascular and respiratory neural control (118) The largest abnormality in neurotransmission currently known in the SIDS population is the binding properties of the serotonergic neuronal populatio n of the brainstem (120) This system like those changed after PNE plays an extensive role in maintaining homeostasis during hypoxia. It is thought than t hat hypoxia may be the most vulnerable stressor to SIDS infants. Neuronal populations critical in infant respiratory function have even demonstrated striking gender differences which could be related to the increased number of deaths seen in male s (141) The increased incidence of SIDS in male children born to smokers has been theorized to then be related to critical changes in cardiovascular o r respiratory control circuits within the brain and/or a change in the normal development of coordinated integration of the two systems (177) Indeed all current evidence suggests that SIDS is a multi -factorial syndrome (82) In the ear ly 1990s, rodent studies were the first to demonstrate a real link between the role of PNE and an increased mortality in response to a hypoxic challenge (216) Since then much of the work on PNE has focused on changes in respiratory control either during eupneic breathing or during acute hypoxic events. In general these studies identified some specific developmental changes in respiratory control that occur following PNE. For example, although no change in eupenic minute ventilation is seen at any postnatal day (10, 96) PNE has been shown to induce developmental changes in respiratory frequency at postnatal day (P)10, and tidal volume starting around P14 (96) Additionally, during acute exposure to hypoxia, PNE pups at P7-8 have been shown to ha ve a significantly elevated respiratory frequency compared to controls

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48 during the initial (dynamic) response and at steady state (10 minutes after a continuous hypoxic exposure) (10, 143) Increases in tidal volume during hypoxia have even been reported to be sustained out to P21 (143) In addition to changes in respiratory patterning, several studies have documented severe bradycardias in SIDS victims, prior to (77, 113, 149, 183) and during the terminal SIDS event (183) Additionally, analysis of heart rate variability (HRV) analysis has demonstrated significant differences in SIDS victims compared to normal infants, at least as a function of sleep state. These differences include both a reduced beat -to -beat variability (203) and a reduction in the high frequency spectral power, thought to be related to changes in parasympathetic drive (64) Interestingly, these differences have been shown to demonstrate gender differences (85) again supporting the theory that SIDS strikes differently in boys and girls. In both rodents and lambs, the heart rate (HR) response to hypoxia has been shown to be blunted during the first few postnatal days (P1 -2) follow ing PNE compared to nonexposed controls, (83, 219) Surprising, however, the basal HR data has been slightly conflicting, although this is probably a function of the developmental time p eriod evaluated or species used (59, 83, 219) These cardiovascular data suggest a potential problem in autonomic function throughout at least the first week in rodent life, predisposing affected offspring to potentially life -threatening bradycardias. Thus, based on the information discussed above, the revised hypothesis of mechanisms underlying SIDS now includes both inappropriate respiratory and cardiovascular responses to at least one hypoxic episode (165)

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49 One potential indicator of appropriately integrated physiological function between the respiratory and cardiovascular systems is the generation of respiratory sinus arrhythmia (RSA). RSA is marked by an acceleration of HR during inspiration and a subsequent slowing during expiration (24) In adults, RSA can be observed during ea ch respiratory cycle and is particularly prominent when a person is at rest. Abnormalities in RSA are associated with pathophysiology in both neonates and adults and also increased mortality (32, 46, 149, 224) The phenomena of RSA partly reflected the central integration of pulmonary stretch receptor activity as well as the mechanical effect of lung inflation on venous return and cardiac stretch (74) RSA generation, however, also involves the central neural integration between the central respiratory neural pattern generator and parasympathetic nervous system (6, 24, 241) For example, in vitro e xperiments using P4-10 brain slices have confirmed the presence of neural network driven changes in vagal parasympathetic motoneuron output that are specifically timed to respiratory motor output. Moreover, there is now in vitro evidence that PNE alters normal inspiratory related neurotransmission to vagal motoneurons both during normoxia and hypoxia (57, 97, 98, 170, 171) More specifically, during hypoxia/hypercapnia, PNE causes both an accelerated decrease in inhibitory neurotransmis sion and a recruitment of an excitatory pathway (108) potentially predisposing an individual to excessive bradycardias. A mature, or adult like, RSA pattern is not established until late in the postnatal development of rodents (2, 3, 114, 208, 238) Thus, when considering the potential multi -factorial impact of PNE on cardiorespiratory integrati on it may be important to evaluate system function beyond the first ten days of life (the oldest time point currently

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50 evaluated in vitro ). Furthermore, new evidence suggests that the majority of the respiratory abnormalities seen with PNE primarily occur w ithin a select postnatal time window, often later than the first week (~P718) (96, 143) Therefore, an examination of the developmental changes in cardiorespiratory integration beyond P10 in PNE rodents compared to controls is needed. This information may provide new insight in the current understanding of how PNE relates to SIDS. This may be particularly relevant since it is estimated that the first week of a rodents life may actually corresponds to the last trimester in humans (186) The current study was undertaken to evaluate changes in cardiorespiratory in tegration (HRV) that occur beyond P10 in response to PNE. Additionally, since male infants are more likely to succumb to SIDS than their female counterparts (158) and few studies in the rodent model have examined the role of sex in cardiorespiratory development, both male and female pups were evaluated. It was hypothesized that, although no differences between PNE and salineexposed pups would exist during overall resting HR and RR as a function of age, developmental differences in both resting RSA and cardio-respiratory responses to hypoxia would exist in the second week of life (P13 and 16) of PNE pups and these changes would be most dramatic in male offspring. Methods All experiments were performed on Sprague Dawley rats (SD; Harlan Industries, Minneapolis, IN ) housed in the University of Florida (UF) animal care facility. All procedures were approved by the UF Institutional Animal Care and Use Committee (IACUC) prior to use. All rats wer e maintained under standard environmental conditions (12:12h light:dark cycle) with free access to food and water.

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51 Prenatal Nicotine Exposure Rat dams (2315g) were implant ed with osmotic mini pumps (Durect Corporation Alzet Osmotic Pumps, Cupertino, CA) o n day 6 of gestation (of a normal 22 day gestation period). Dam s were briefly anesthetized to a surgical plane with isoflurane (5% initially, then 2 -3% for the remainder of the surgery) and a small i ncision was made between the scapulae. A 28 day osmotic mini -pump (2ML4 Alzet) was inserted subcutaneously which allow ed for 24 hour infusion of either nicotine tartrate ( PNE, n=16: 6mg/kg/day of nicotine diluted in saline) or vehicle ( CON, n=10: 2mL of saline) (168) The nicotine dose was based on an estimated mid pregnancy weight gain of 54% gain from the initial weight taken at the time of pump implantat ion (average weight gain for a full term pregnancy was determined in preliminary studies of dams without pumps, n=8). Following subcutaneous positioning of the pump, the incision was closed and d am s were weaned from isoflourane (2.5% -0%) and allowed to awaken naturally from anesthesia Following a recover y period the dam s were returned to their individual cage s and observed daily until parturition. The day of parturition was termed P0. On P3, pup sex was determined, and litters were culled to 8 pups (4 fem ales: 4 males when possible) to ensure proper nutrition for all pups. Pups were weaned on P21 and housed in pairs. All pups remained with the litter or littermates until the day they were selected for the study, P13, P16 or P26. Rat Pup I nstrumentation O n P12 14, or 24, pups were retrieved from their home cages and anesthetized to a surgical plane with isoflurane gas ( 5% in 100% O2 with a flow rate of 0.6 mL/min initially, then 2.5 3% for the remainder of the surgery) Following the induction of anesthes ia, the h air on the nape of the neck was shaved and t he area was disinfect ed.

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52 A small incision was then made along the midline of the ventral surface of the neck to allow for the insertion of t wo thin wire electrodes with barred tips (Teflon coated; 0.005 mm diameter, A M Systems Inc., Carlsborg, WA) The wires were placed subcutaneously directed toward f ront limbs for electrocardiogram recording (ECG: Lead I). Another thin wire electrode was placed subcutaneously along the ventral surface of the back to serve as a ground wire The incision was closed with tissue glue (Nexaband S/C, Abbott Laboratories, Chicago, IL) and sutures. For P12 and P14 pups, all three electrodes were connected to a back mounted pedestal (E363BM PlasticsOne Inc., Roanoke, VA ) th at was secure d in place with suture just on top of the shoulder blades For pups instrumented at P24 pups, the ECG and ground wires were secured into a head mounted pedestal (MS363 PlasticsOne Inc) that was secured to the skull with dental cement (Ortho -Jet, Lang Dental, Wheeling, I L) and the insertion of two small jewelers screws (00 96x1 /16 PlasticsOne Inc.) into the skull. Following instrumentation, all incisions were closed, pups were weaned from isoflourane (2.5% 0%) and allowed to naturally awak en. Animal were then allowed to recover on a warming blanket Post surgery all pups receive d subcutaneous fluids, analgesics (Buprenorphine, 0.01m g/kg ), and topical antibiotic cream (Alpharma, Baltimore, MD). Animal were then allowed to recover on a warmi ng blanket D uring recovery, the P12 and P14 pups were fed every half and hour with a milk substitute (30% fat milk -based cream) Once able to right themselves the pups were returned to the mother and continually monitored until they beg an feeding on their own and the mother returned to grooming them

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53 Data Collection On the day of recording, pups were removed from their home cages Each pups pedestal was connected to a single brush 6 channel commutator (SL6C/SB PlasticsOne Inc.) via a tether system allow ing for unrestricte d movement during the recording session. The animal and tether w ere placed in a sealed plexiglass plethysmograph chamber (Buxco Electronic Inc, Wilmington, NC) with room air ( 21% O2) flowing at a rate of 2mL/min. The plethysmograph was fitted with a pressure transducer (TRD5700 Buxco Electronic Inc) to allow monitoring of respiratory related pressure changes and non-invasive recordings of respiratory rate (RR). Next the plethysmograph was placed in an electrically shield ed copper cage. A thin wire thermister was inserted into one of the airflow holes in the plethysmograph and chamber temperature was continuously monitored. N ormal ambient temperature wa s maintained in the plethymograph wit h heat lamps to the temperature appropriate for each age (P1 3: 3334oC P16: 2930oC and P25:22oC) All experiments occurred between 1230 and 1700 h. For all age groups, output from the single brush commutator directed the ECG signal to Grass preamplifier (P511, Grass Instruments, West Warwick, RI), w h ere the signal was amplified and filtered. The ECG signal and the output from the plethysmograph pressure transducer were then sent through a real time data acquisition interface (Power1404, Cambridge Electronics Design (CED), Cambridge, UK) to a PC comp ut er equipped with complimenta ry data analysis software sys tem (Spike2, CED, Cambridge, UK). Each pup was acclimated to the plethysmograph for at least 20 mins. After acclimation, 20 minutes of baseline recording was taken. After baseline, inflow air

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54 throu g h the chamber was switched from room air to 10% O2, thereby exposing animals to progressively decreasing oxygen levels which reached a minimum of 10%. P13 pups were exposed to 5 minutes of hypoxia and P16 and P 26 pups were exp osed to 7 minutes of hypoxia. Hypoxia levels within the chamber were monitored and recorded with an oxygen monitor attached a port which allowed for the monitoring of oxygen concentration of the air exiting the c ha mber ( AX 300, Teledyne Analytical Instruments, Industry, CA). In the sm aller chambers (P13 and P16), it took ~2 mins for the exiting O2 to reach 10%. In the larger chambers (P26), it took ~4 mins for the exiting O2 to reach 10%. Following completion of the experiment the animals were returned to their home cages and subsequently euthanized with an overdose of pentobarbital (200 mg/kg, i.p.). To avoid a bias in data collection, all male data represent only one male per litter. In females this was also true 79% of the time. No more t han 2 animals were ever used per litter. Dat a Analysis All data were analyzed off -line using Spike 2 software (Cambridge Electronic D e sign). Before each hypoxic exposure, 60 seconds of stable baseline recordings were identified. Baseline HR was determined by the time interval between R -wave peaks in the ECG signal. Respiratory rate (RR) was determined from the time interval between pressure troughs, which represent peak inspiration, in the plethysmograph tracing. During hypoxia, data were selected for analysis only after the O2 content in the plethy smographs chamber dropped to 13%. Data were then averaged from this point in 60 second bins until either the end of the recording period or 5 minutes of data had been collected. To evaluate changes in HR and RR, each animals HR and RR during

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55 each minute in hypoxia were then compared to the baseline HR and RR values calculated prior to the onset of hypoxia. HRV analysis was performed on two separate periods or segments of quiet baseline. Each baseline segment included 2 min. of consecutive recordings of HR. Each segment of the derived recordings was converted to a tachogram, a record of the timing between RR intervals (RRI). The tachogram was then imported into a HRV software program (Biosignal Analysis Group, Univeristy of Kuopio, Finland) for both linear and non-linear analysis of data. Next, the tachogram was interpolated at 10 Hz and detrended via the smoothness priors formulation (alpha=1000) (172, 232) The autoregressive model was set to the 40th order and the Welchs Periodogram window width was designated to 512 points with an overlap of 256 points in the Hanning window. Following analysis, both the power in the high frequency (H F) range and the location of the HF peak were documented. The power of the low frequency (LF) range was used to calculate a LF to HF ratio or LF/HF, an index of sympathovagal balance. To evaluate developmental changes in cardiorespiratory integration, th e correlation between the location of the HF peak identified through HRV and respiratory rate were used as an index of RSA (175) To ensure that enoug h data was available to provide a reliable correlation analysis, two separate 2 min. periods from each animal was evaluated. To determine significant differences in baseline HR, RR and HRV between sexes at different developmental time points, a two way AN OVA (factor 1: sex and factor 2: postnatal day) was used. To identify specific effects of PNE on development, a one way ANOVA was used to examine the change in HR and RR as a function of CON groups

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56 HR and RR. To evaluation the relationship between the loc ation of HF peak of HRV analysis and RR, as a measure of RSA (17, 91) linear regression analysis was performed. A one way repeated measures ANOVA was used to determine significance differences between PNE and CON groups in both the change in HR and RR under hypoxic conditions. When appropriate, a Scheffes post -hoc test was used to assess differences between treatment groups across the different developmental ages A Bonferroni adjusted t -test was used to determine pvalues as a function of oxygen concentration. When a t -test was run, we only examined the relationship of HR and RR at a specific oxygen concentration compared to the first minute of the hypoxic exposure. Pvalue of 0.05 was accepted for significance. All data are presented as meansSEM. Results Baseline Physiology As shown in Table 2 1, weight significantly increased with age (p <0.0001). However, no differences in weight were found between treatment groups in either males or females. Figure 2 1 A show typical recordings from two separate CON male pups, at P13 and P26. These exampl es illustrate two main developmental changes that were observed in both treatment groups. First, as the animals aged there was general slowing of RR. Second, the maturati on of RSA is also represented in the associated HRV analysis ( Fig. 2 -1 B) by the incr ease in the power of the HF range with developmental maturation and more apparent breath to breath fluctuations in HR Comparisons between the average baseline HR and RR at each developmental time point and for each treatment group are shown in Figure 2 2 On average, in the CON pups there was no significant difference in baseline HR or RR between males and females across development (Figure 2 -2A). Similarly, PNE pups demonstrated no

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57 significant sex differences in HR with age (Figure 2 2B: upper panel). A two way analysis of variance, however, did identify a trend for an effect of postnatal d ay on baseline HR (P=0.07 7 ). A significant effect of postnatal day (P=0.012) was identified for mean baseline RR in the PNE animals but no significant difference between males and females was found (Figure 2 -2B: lower panel ). Post -hoc analysis of baseline RR in the PNE animals, independent of sex, demonstrat ed that the RR at P26 was significantly lower compared to the RR at P16 (P=0.013). To further evaluate the effect o f PNE, the change in HR and RR for each PNE pup as a function of the average HR and RR of the CON group at the same developmental time point for each sex was examined (Figure 2 3 ). For male PNE pups (Figure 2 -3: left panels), a one way analysis of variance demonstrated that postnatal day signifi cantly affecte d both HR and RR (P=0.01 3 and 0.010 respectively). P ost hoc analysis identified that the average change in PNE male HR relative to CON was significantly elevated at both P13 and P16 compared to P26 (P= 0.00 3 and 0.016 respectively). Additionally, the average change in RR in the PNE pups was significantly elevated at P16 compared to P26 (P=0.013) and tended to be significantly higher than P13 (P=0.0674). In contrast, the female PNE pups demonstrated no s ignificant effect of postnatal day in either HR or RR change from CON (Figure 2 3: right panels). RSA Figure 2 1B illustrates the effect of age on the development of RSA as demonstrated by HRV analysis in the CON male pups. As shown in the left panel, alt hough there was a peak in the HF range (red line) identified at P13, the power in the HF range was approximately equal to the LF peaks power. At P16, the power in the HF

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58 range increased, relative to P13, but the power in the LF range also increased. Final ly, at P26, the power in the HF range increased and now the HF power was greater than that observed in the LF range, reflecting more of an adult pattern of HRV. Figure 2 1B also illustrates the normal shift in the location of HF peak on the x axis (HF loca tion) as a function of age. In adults, the location of the HF peak reflects respiratory rate (17, 91) The mean power for the HF component and the LF as a function of the HF power (LF/HF ratio) is shown in Figure 4. For all developmental ages, the presence of the high frequency (HF) compone nt was identified in both males and females regardless of treatment (Figure 2 -4: upper panel s ). No significant differences existed in the power of the HF or LF/HF ratio in males regardless of treatment (Figure 2 -4: left panels). However, females did demonstrate a significant effect of postnatal day on HF power (P=0.0106; Figure 2 -4: top right panel). Post hoc analysis demonstrated that the mean power in the HF range at P13 was significantly lower than the HF power at P26 (P=0.0067) and tended to be lower t han the HF power at P16 (P=0.0677). Like males, females demonstrated no significant differences between treatment groups or postnatal day in LF/HF (Figure 2 4: bottom right panel). To evaluate the development of RSA, the location of the HF component (depic ted in Figure 2 -1B: left panel) was correlated with each animals corresponding baseline RR (Figure 2 5) (175) Male development of RSA is depicted in the left panels of Figure 2 5. The upper left panel demonstrates that in both CON and PNE male pups at P13, the correlation coefficients were small (R2=0.03 and 0.05 respectively; Table 2 2). By P16, in both CON and PNE males, the HF location and RR were well correlated (R2=0.84

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59 and 0.85 respectively; Table 2 -2). Yet, by P26, PNE male pups demonstrated only half the correlation coefficient of CON pups (R2=0.43 vs. 0.81; Table 2 2). The development of RSA in female pups is depicted in the right panels of F igure 2 5. Similar to the male pups, at P13 CON females demonstrated a weak correlation between RR and the HF peak location (R2=0.005; Table 2 -2). Paradoxically, PNE females at P13 demonstrated a strong correlation (R2=0.90; Table 2 2). By P16, CON females increased the correlation between the two factors (R2=0.56), but the strength of the correlation was still less than the PNE females (R2=0.72). Finally, at P26 both CON and PNE females demonstrated high R2 values (R2=0.93 and 0.91; Table 2 -2). Development of RSA was also characterized by the slope of regression lines (Table 2 2). The slope of the regression line in CON males at P13 was not significantly different from zero, nor was the slope for CON females at this time. However, CON males demonstrated a negative slope whereas the slope of females was slightly positive (-0.1603 and 0.04115 respectively). By P16, all CON animals demonstrated slopes which were significantly different from zero, but did not reach 1. Similarly at P26, all CON pups had slopes that were nearing 1. In PNE male pups at P13, the slope of the regression line was similar to CON males ( 0.1317). However, PNE females at P13 demonstrated a slope which was positive and greater than 1. By P16, PNE male pups now demonstrated a positive slope which was also greater than one. However, the slope of PNE female pups at P16 had decreased (0.5393), though it too was significantly different from zero. In P26 pups, both PNE males and females demonstrated slopes which were positive and close to one.

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60 Postnatal C hanges in R esponse to H ypoxia The typical response to hypoxia, at P13 in a CON and PNE male pup is shown in Figure 2 6. In the CON pups at P13, HR rose significant higher than PNE pups at the same age. No differences were seen in the mean rise in RR under hypoxia at this developmental time point, but the PNE appeared to have less variability in RR and tidal volume. The average change in HR in response to hypoxia as a function of postnatal development in CON versus PNE male pups is demonstrated in the left panel in Figure 2 -7. At P13, a repeatedmeasure analysis of variance found a significant effect of level of hypoxia on HR ( P=0.014; Figure 2 7: top left panel). Post hoc analysis identified a significant increase in the change HR at 11% O2 an d the first minute at 10% compared to the minute average at 12% in all males (P=0.007 and 0.007 respectively). Additionally, the change in HR in the PNE male pups in response to hypoxia was significantly blunted compared to the male CON pups (P=0.008), wit hout a significant interaction with percent O2. At P16, males showed no significant change in HR as a function of the change in the percent O2 nor did CON pups differ from PNE pups (P>0.05; Figure 2 -7: middle left panel). At P26, no significant independent effect of treatment or O2 level were found, but an interaction between these variables was present (P<0.0001). Although CON animals did elevated HR during hypoxia, they did not significantly change their HR throughout the hypoxic exposure (Figure 2 -7: bottom left panel). Paradoxically, PNE animals demonstrated a significant increase in their HR during the time spent at 11% O2 compared to the first minute spent at 13% O2 (P=0.0056 and 0.012). More importantly, mean HR in the PNE pups were significantly

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61 hig her than CON animals during the last three minutes of the hypoxic exposure (P=0.044, 0.021 and 0.016 respectively). The development of the HR response to hypoxia in female pups is shown in the right panels in Figure 2 7. Although there were no significant difference between CON or PNE females in the HR response at P13 (Figure 2 7: top right panel), there was a significant effect of the level of O2 on HR (P=0.0189). Further analysis identified that at P13, both groups of females increased HR during the min ute spent at 11% compared to the minute spent at 12% O2 (P=0.0040). At P16, females did not demonstrate a treatment effect either (Figure 2 -7: middle right panel), but all female animals significantly increased HR at both the minute spent at 11% and at 10% O2 from the minute spent at 12% (P=0.0001 and 0.0006). The bottom right panel of Figure 2 7 demonstrates that no differences were found in the HR response to hypoxia between treatment groups or O2 levels at P26 in females. Figure 2 8 demonstrates the developmental changes in the respiratory response to hypoxia. At no developmental time point was a treatment effect found in either the males or females. In the top left panel of Figure 2 8, all male pups (regardless of treatment) significantly increased their RR from the minute averaged under 12% O2 to that under 11%. By P16 (Figure 2 -8: middle left panel) all males combined demonstrated a significant effect of O2 levels (P=0.0052). Post hoc analysis however, found no differences from the minute spent at 12% t o any of the other times (P>0.0125). At P26, no differences were seen in RR response in male pups (Figure 2 -8: bottom left panel).

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62 Similar to males, no treatment effects were detected in the RR response in female animals across development (Figure 2 8 righ t panel). Additionally at P13, no significant differences were found as a function of O2 concentrations (Figure 2 -8: top right panel). At P16, however, RR in the female pups did demonstrate a significant effect of hy poxia level (P<0.0001; Figure 2 8: middl e right panel). P16 female pups significantly dropped their RR during all three minutes spent at 10% O2 compared to the time spent in 12% (P=0.0119, 0.0088 and 0.0012). No differences were observed in RR response throughout the hypoxia exposure at P26 in t he female pups (Figure 2 -8: bottom right panel). Discussion The present study was undertaken to evaluate the impact of PNE on cardiorespiratory integration between P13 and 26 with the added consideration of the contribution of sex. In CON animals, no signi ficant changes in resting HR or RR were identified between the sexes or across the age groups evaluated. The only significant differences were identified using HRV analysis First, in the CON females HRV analysis d emonstrated a significantly lower HF pow er in the CON females at P13 compared to P2 6 A similar reduction in the HF power during early development w as not observed in the CON males Second, evidence of the presence of RSA was not identified until P16 and this relationship was similar in both sex es and was not completely mature until P26. When compared to their age and sex matched cohorts, several striking cardiorespiratory changes associated with PNE were identified. First, only PNE male offspring demonstrated significant differences in resting HR and RR at P16 compared to CON males. Second, PNE induced premature development of RSA that was associated with an elevated slope of the HF peak -RR relationship and this abnormality occurred at

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63 different time points dependent upon the sex of the animal. Finally, only the HR response to hypoxia was significantly altered in PNE male offspring. Alterations included a blunted response at P13 and an exaggerated response at P26. These observations support our original hypothesis that the impact of PNE would have a more dramatic effect on males and uncover the sex -based developmental differences in HR maturation. Sex D ifferences in D evelopment of HR in CONs Previous studies investigating the normal development of HR in the neonatal rat have demonstrated that, in general, HR increases with age, although the most dramatic changes occur between birth and P10-13 Beyond P13, the parasympathetic system begins to integrate with the sympathetic system and plays a more prominent role in resting HR (114, 115) In the present study no sig nificant changes in HR were identified between P13 -26 in either the CON males or females. The CON males however tended to increase their resting HR from P13 to P16 and then maintained similar HRs from P16 to P26. In contrast, resting HR in CON females chan ged very little during any of the developmental time points studied. Our observation that sex does not play a significant role in resting HR development between P1326 is supported by p revious studies of normal cardiovascular development that have either n ot found sex based differences (114, 115, 138) or not evaluate potential sex differences (57, 95, 171) Thus, it is possible that if sex differences in HR development exist in the rodent, these differences are very subtle. In the present study HRV analysis was used to further evaluate subtle sex -based differences in autonomic development. In the CON females a significant increase in HF power from P13 to P16 was identified, but there were no corresponding changes in the

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64 LF/HF ratio. This result might be expected since tonic parasympathetic regul ation of HR in rodents has been reported to begin ar ound P15 and incr ease out to ~ P21 (2, 114) The absence of a parall el change in baseline HR during this period of increasing parasympathetic drive has been demonstrated by others (114) This suggests that there may also be developmental changes occurring in intrinsic cardiac pacemaker activity during this time point. Indeed, there is some evidence that the intrinsic rate of pacemaker cells in the sinoatrial node increases throughout this time period examined in the present study (237) Unfortunately HRV analysis does not identify changes in intrinsic HR (106) Similar changes in HF power or LF/HF ratio between P13 to P16 were not identified in the CON male pups. Indeed, the HF power at P13 was very simi lar to the power at P26, despite a noticeably lower HR at P13. This suggests perhaps that males, in general, may develop parasympathetic drive sooner than females (as demonstrated by the HF power). The increase in HR in the males that was identified to o ccur from P13 to P16 then may be related to the above mentioned changes in intrinsic pacemaker activity. One possible mechanism for sex based differences in parasympathetic function m ay be related to fluctuations in hormones that occur during early postna tal life. Male neonate rodents, and humans alike, experience a large surge of testosterone during the immediate postnatal period (63, 99, 128) It had been hypothesized that this surge is responsible for the mascularization of the brain (78, 101) and epidemiological evidence suggests that fund amental differences must exist between male and female neural development since developmental diseases, such autism (185) and general mental retardation (25) tend to strike in the young male population more prominently

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65 than females. Taken together, fundamental sex differences may indeed exist in developmental timing of brain processes, and possibly the autonomic nervous system specifically. Sex D ifferences in D evelopment of RR in CON s Previous studies have identified a developmental peak in resting RR that occurs sometime during the second week of rodent life. However, the exact time course of peak RR development has been somewhat conflicting (96, 134) In the present study however we did not identify any significant changes in RR across development or between the sexes, although there was a more noticeable drop in RR from P13 to P16 in the males. It is possible then that previous disagreements in the exact timing of this peak in RR development may be a function of the behavioral state, since it is known that RR is highly influenced by sleepwake state in both adults and infants (38, 130, 166) Though care was taken in the present study to minimize th e variability of data by selecting periods of quiet baseline recordings, sleep/wake state was not directly exami ned. Indeed in human infants, males have been reported to have faster RR s during the first sixth months of age during all sleep states compared to females (93) ; and in 3140 year olds, normal male subjects displayed more sleep -related breathing disturbances than female counterparts (187) Sex D ifferences in D evelopment of RSA in CON pups To examine the integration of cardiovascular and respiratory control in normal development, correlations between the location of the HF peak and RR were evaluated. In both CON males and CON females at P13, R values were small and slopes were not significantly different from zero, suggesting that al though HF power is present (from HRV data) respiratory modulation of the HF peak is minimal. By P16, RR and HF

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66 location were better correlated in both sexes, suggested by the increase in both R values and slopes which become significantly different from zero. By P26, both CON males and females exhibited a strong relationship between HF peak location and RR. Moreover, the slope of the relationship approached unity. These correlations demonstrate that respiratory modulation of parasympathetic drive develops throughout the postnatal period starting around P1 6 and are fully developed by P26. Furthermore, the observation that respiratory integration wi th the autonomic nervous demonstrates no sex based differences is interesting since our cardiovascular data analysis suggested males developed parasympathetic tone (HF power) earlier than females. Indeed, based on the cardiovascular results alone it might have been predicted that males would develop RSA earlier than females. Yet since this did not occur, it appears that the integration of the two systems depends on precise developmental timing of the two separate systems. Alterations in the developmental time course of one or both systems, such as might occur following PNE, could leave the cardio-respiratory system temporarily unstable. Sex D ifferences in the R esponse to H ypoxia in CONs Alt hough the parasympathetic system does not play a major role in r esting HR during early postnatal development, it can be recruited during hypoxia. For example, in a recent study by Fewell and colleagues, cervical vagotomy was shown not to alter baseline HR at P1 -P2 or P10 -P 11 in rodent (60) During 5 min of hypoxia however, HR are significantly elevated in vagotomized animals compared to vagal intact pups. In the present study a hypoxic stimulus was used to examine how HR and RR respond to a physiologic challenge between P1 3 -P 26. At all postnatal time points, both males and females demonstrated the same pattern in HR and RR regulation, including an initial

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67 increase in rate, until the chamber reached oxygen levels between 11-12%, followed by a decline in rate. This pattern e ven occurred at P13, when there was little evidence of RSA-based cardiorespiratory integration in either sex and evidence from HRV analysis that parasympathetic drive was significantly reduced at this time point in the females. Together, these data sugges t that despite fluctuations in parasympathetic tone at different developmental periods, the capacity to induce cardiorespiratory integration during physiological challenges exist at all postnatal time points in both males and females. I mpact of PNE on S ex B ased D ifferences in C ardiorespiratory D evelopment Although no major differences in cardiorespiratory function between the sexes were identified in the CON animals, PNE appeared to affect postnatal development of HR in a sex dependent manner. F or example male PNE pups expressed a significant elevation in baseline HR relative to CON males at both P13 and P16. In contrast, PNE tended to decrease HR in the females at P13 and P26, although these changes were not significant. Identification of sex dependent effects on HR following PNE may provide an answer to previous conflicting reports on PNEs effect on basal HR. For example, some studi es ha ve reported that PNE may induce a reduct ion in HR (59) while other studies have reported an increase in HR (83) or no change (219) Inde ed, a study that evaluated equal number of males and females did not identify any effect of PNE on resting HR, a finding that might be expected if males demonstrated an increase and females demonstrate a decrease in basal HR following PNE exposure. The res ults of our study strongly suggest that it is important to consider sex when examining the effects of PNE.

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68 Further analysis of the effect of PNE on autonomic function through HRV analysis demonstrated no significant differences from CONs. It should be not ed however that PNE females at P13 demonstrated a lower LF/HF ratio, an indicator of reduced sympathetic tone. A decrease in overall sympathetic drive in the PNE females could explain the generally lower basal HR in the PNE females compared to CON females at P13. Indeed, the basal HR of the PNE females appeared more similar to the HR of the CON males at this time point. This observation raises the possibility that one effect of PNE may be to masculinize female autonomic development. Work done in humans demonstrated that nicotine alone inhibits aromatase activity in the placenta (11) Hormone production during pregnancy involves an intricate play between all parts of the maternal -placental -fetal unit. Since feminization of the brain occurs in utero, it is possible that a reduction in available estrogen levels and increase in testosterone (due to a reduced aromatase activity) may serve as a masculinizing event Accordingly, PNE has been shown to abolish sex differences in sac charin preferences in adu lt rats (132) and correlate with a mascularized/defeminized sexual orientation in female offspring (52) The observation that the effect of PNE on HR was only significant in males was not surpri sing since PNE is a significant risk factor for SIDS and there is a higher incidence of SIDS in males (142, 158) Accordingly, in the present study only m ale offspring demonstrated significant changes in basal RR following PNE compared to CONs. In this instance, a significant increase in RR in the PNE relative to CON males occurred selectively at P16. Evaluation of both HR and RR in male offspring, sugges ts that PNEs influence on cardiorespiratory integration occurs at a very specific

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69 developmental time point around P16. As previously mentioned this time point represents a period of central integration of the parasympathetic system. As such, a shift in the timing of development or a change in neurotransmitter or receptor expression in either autonomic or respiratory central control systems induced by PNE may temporarily leave the system unstable during state dependent transitions or physiological challenges which may trigger a severe apnea or bradycardic response. Examination of PNEs effect on respiratory related cardiovascular function, or RSA, illustrated some intriguing alterations. At P13 PNE males demonstrated similar R values and slopes compared to CON males, yet by P16, PNE males demonstrated a slope greater than one for the relationship between HF peak location and RR, suggesting a premature development of complete, and possibly over development, RSA compared to CONs. The slope of this relationship returned to values similar to the CON males at P26. In contrast, the female PNE pups demonstrated a RSA slope greater than 1 at P13. However, by P16, the slope of this relationship was not noticeably different fro m their CON counterparts. These dat a suggest that PNE may have accelerated development of RSA, regardless of changes in HF power. This premature development of RSA occurred at different time points in males and females, supporting the importance of evaluating sex -based di fferences. Furthe rmore, PNE may have induced an over development of RSA (a slope > 1), possibly reflecting a period of system instability. In support of this theory, work done in vitro has demonstrated that PNE creates greater inspiratory related GABA activity in the car diac vagal neurons even during baseline conditions (171) Interestingly, our results suggest that PNE females have resolved this developmental change in cardiorespiratory integration by the time

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70 males pups are beginning to present with it. This observation raises the possibility that females who are compromised by PNE may transition out of the SIDS critical developmental window much faster than their male counterparts, which contributes to their lower death rates The mechanism(s) underlying sex -based plasticity is currently unknown but has been reported to be present following other perinatal insults such as early postnatal hypoxia, in that the adult response to subsequent exposure to hypoxia is unaffected in female offspring but markedly blunted in males. (14) Thus, it appears that female PNE pups can adapt better to PNEs deleterious effects than their male counterparts. Moreover, because evidence of greater developmental plasticity in females in the present study occurred prior to the onset of puberty, it is likely that the potential for greater plasticity in females is not exclusively estrogen based. It shou ld also be noted that both PNE groups significantly dropped RR from P16 to P26. A similar effect was not identified in the CON groups. The overall decrease in RR may be related to a change in overall behavior at this time point. For example, previous rep orts have demonstrated that PNE accelerates the development of circadian rhythms and consolidated sleep patterns (68) Therefore, perhaps PNE pups at P26 had a lower RR because during the recording period they spent more time in NRE M sleep compared to their CON counterparts. To our knowledge however, changes in RR that occur during sleep following PNE have not be previously investigated in rodents. I mpact of PNE on C ardiorespiratory R esponses to H ypoxia The present study demonstrat ed that PNE caused a significant blunting of the HR response to hypoxia at P13, but this effect only occurred in the male offspring. This observation reinforces previous research which has demonstrated the PNE blunts autonomic responses to hypoxia (83, 219) However, the present study is the first to

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71 show that this effect is sex dependent and occurs beyond of the first 10 days of life in rodents. These data further support the hypothesis that females may have greater intrinsic plasticity and are able to better compensate for the physiological abnormalities associated with PNE when exposed to physiological stress. Our findings also are the first to demonstrate that the blunted HR response in males appears to be resolved by P16. The respiratory response during hypoxia however did not demonstrate an y remarkable difference between CON and PNE animal or between males or females. The lack of a developmental effect on RR is somewhat conflicting in the current literature. Some studies have demonstrated significant increases in hypoxic minute ventilation (143) while others have found no changes following PNE (10) However, the effect of PNE on respiration appears to be primarily related to changes in tidal volume. Accordingly, neither of the studies mentioned above identified significant changes in R R during hypoxia. Therefore, the current study confirms the previous finding, as we found no significant effect of PNE on RR responses under hypoxic conditions. Finally, one unexpected result of the current research was that at P26 significant differences in the HR response to hypoxia was identified between PNE and CON males. The PNE males expressed a progressive rise in HR as oxygen levels fell while the CON animal initially increased their HR followed by a decline. This significant elevation in HR durin g hypoxia by the PNE males was paralleled by some indication of a slightly higher LF/HF ratio compared to CON at P26 and lower R2 value for the correlation between RR and HF peak location at P26 compared to both CON males and both females groups. Although this was not original l y predicted, these results should not have been

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72 surprising. First, a recent study has demonstrated that PNE causes cholinergic hypo activity within the brain even into adol escence and that this change was more prominent in males than females (217) Additionally, e pidemiological data suggests that infants exposed to PNE have a greater tendency to develop both hyperactivity and attention deficient disorders (43, 55) reduced memory and spatial ability (35) and increased incid ences of cigarette usages (174) as they get older. Some of these longer lasting effects of PNE have also shown sex differences (54, 174) Although, the majority of previous reports have been on the cognitive function after PNE, only a few studies have examined the impact of PNE on the development of overall cardiovascular health and development of function. Indeed, one study has demonstrated that a PNE associated reduction i n cardiac adrenergic sensitivity persists even into adulthood (27) an d another has identified an angiotensin II dependent hyper -responsiveness that occurs only in male PNE offspring (247) Thus, indicators of elevated sympathetic drive identified in the present study may be mediated by chronic changes in central sympathetic control elicited either as a compensatory response to reduced cardiac sensitivity or abnormalities in the brain angiotensin system. Of particular importance then is the potential for PNE to predispose male offspring to an elevated incidence of cardiovascular disease in later life (13) Methodical Considerations The largest consideration for the present study was the time allowed for recovery after surgery. The pups evaluated i n the present study were chronic instrumented to allow for a more realistic evaluation of HR regulation in the conscious unrestrained animals. Because difficulties in pup weight gain were identified when instrumentation took place prior to P12, animals re corded at P13 were only allowed one day of recovery.

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73 Although both treatment groups received surgery at the same developmental time point, there is some evidence to suggest that prenatal stress affects the responsiveness of the hypothalamic -pituitary adren al axis. Thus, it is possible that some of the alterations seen as a result of PNE were due to different responses to the stress of surgery. However, we tried to minimize all stress associated with instrumentation by making sure that the pups were returned to their home cage and received maternal care following their return. Moreover, any differences in stress responses to surgery may have been minimal since our results are similar to those in other animals models (83) Additionally, surgery recovery time has not been shown to effect developmental changes in autonomic function (51) Summary The present study provides an evaluation of the impact of PNE and sex on cardiorespiratory integration during the se cond and thirds weeks of postnatal development. In general, the development of autonomic nervous system function, both activity and sensitivity, is different between males and females. The present study confirmed previously reported alterations in the hypoxic response after PNE, which support the theory that SIDS is related to abnormal autonomic responses to hypoxia. We also extend this theory to include the idea that SIDS may be related to alterations in the development of parasympathetic sensitivity which is compensated for differently between the sexes (mainly earlier development in PNE females) and abnormalities in the integration of respiration and the autonomic system may be detected by changes in the correlation or slope of the relationship between RR and the HF peak location. The sex differences identified in the present study may account for the gender bias currently present in SIDS mortalities. Furthermore, the effects of PNE on autonomic function in

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74 male offspring were identified to persist into l ater developmental time points than initially thought and may provide new insights into an increased incidence of hypertension in certain adult populations.

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75 Table 2 1. Pup weight Treatment Day Males Females N Weight (g) N Weight (g) CON P13 5 291 4 292 P16 5 332* 4 321* P26 7 703^ 5 64 4^ PNE P13 5 282 4 251 P16 6 362* 6 332* P26 8 703^ 6 611^ All data represented as MeanSEM. represents significant difference from P13 and P26 regardless of treatment, ^ represen ts significant increase from P13 and P16 regardless of treatment.

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76 Figure 21. Representative tracings from chronically instrumented control male rat pups illustrating the development of respiratory sinus arrhythmia (RSA). A) The tracing on the upper left versus right show the maturation of a noticeable fluctuation in heart rate (HR) with each breath (downward pressure change in th e plethysmograph (PG) between P13 and P26. B) Typical changes in individual HRV analysis profiles across development. The frequency profile on the left side is from a male pup at P13, the middle is P 16 and the right side is P26. Note the age-dependent fluctuations in the high (HF) and low (LF) frequency ranges. Also note the leftward shift of the location of the HF peak (identified in the P13 tracing) with age as it corresponds to a developmental slowing of respiratory rate (as shown in panel A).

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77 Figur e 22 T he effect of prenatal nicotine exposure (PNE) on developmental changes in baseline heart rate (HR) and respiratory rate (RR) betwe en male and female offspring. A) Comparison HR and RR between sexes at different developmental time point for pups ex po sed to prenatal saline (CON). B) Comparison HR and RR between sexes at different developmental time point for PNE pups. Males are represented by open shapes and dashed lines. Females are represented by closed shapes and solid lines. # represents a significant drop at P26 compared to P16 independent of sex.

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78 Figure 2 3. Effect of PNE on development changes in baseline heart rate ( HR) and respiratory rate (RR) relative to age and sex matched controls (CON). Male data are represented on the left. Females are represented on the right. PNE associated changes in HR and RR within each age group reflect the change from the average age and sex matched CON. indicates significantly different from P26

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79 Figure 2 4. Impact of PNE on baseline heart rate variability (HRV) componen ts throughout development in both males and females. Males are represented in the left columns and females are on the right. Open bars illustrate mean values from controls (CON) and black bars represent PNE data. The high frequency (HF) power is represent ed as mms2. The low frequency (LF) power is represented as a ratio of the corresponding HF power. represents significant difference from P26.

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80 Figure 25 The effect of PNE on the relationship between the location of the high frequency (HF) component of HRV and respiratory rate (RR). Left columns represent data from male offspring across development and female offspring are represented in the ri ght column. Open shapes and dashed lines show data from control (CON) animals. Closed shapes and solid lines are data PNE offspring.

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81 T able 2 2. Correlational values for RR vs HF location Postnatal Day Sex Slope R 2 CON PNE CON PNE P13 Male 0.1603 0.1 317 0.03 0.05 Female 0.04115 1.283 0.01 0.90 P16 Male 0.3574 1.248 0.84 0.85 Female 0.5322 0.5393 0.56 0.72 P26 Male 0.8705 0.989 0.81 0.43 Female 0.869 0.8742 0.85 0.80

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82 Figure 26. Representative tracings from one CON and one PNE male pup at postnatal day 13 during the first two minutes of an acute hypoxic exposure. A.) The dashed lines represent the trans ition from 12% to 11% O2 w ithin the recording chamber Tracings include heart rate (HR), respiratory rate (RR) and pressure fluctuations associated with respiratory effo rt in the plethysmograph (PG). B) Expanded time scale of upper tracing, illustrating a generally HR response in the PNE animal to hypoxia.

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83 Figure 27. Effect of PNE on sex based h eart rate (HR) response s to lowering oxygen concentrations. Left column represents male data across development. Female data are represented in the right column. Open shapes and dashed lines show average data from control (CON) animals. Closed shapes and solid lines represent PNE animal data. # represents a significant effect of treatment alone (p=0.008). ^ indicates significant differences from 12% within all groups (p 12% within the PNE group alone (p nificant difference between PNE and CON at a given O2 level (p

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84 Figure 28. Effect of PNE on sex based r espiratory rate (RR) response s to lowering oxygen concentrations. Left column represents male data across development. Female data are represented in the right column. Open shapes and dashed lines show average data from control (CON) animals. Closed shapes and solid lines rep resent PNE animal data. ^ indicates significant difference from 12% within all groups (p

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85 CHAPTER 3 IMPACT OF PRENATAL N ICOTINE EXPOSURE ON THE DEVELOPMENT OF SLEEP DEPENDENT CARDIORESPIRATORY FUNCTION Introduction Sudden Infant Death Syndrome (SIDS) is defined as the unexplained death of an otherwise healthy infant, under one year of age, during sleep (246) Although in cidences have declined as a result of the Back To Sleep campaign in 1995, SIDS still accounts for one third of all infant mortalities in the United States (146) Current clinical evidence suggests that SIDS results from an inappropriate cardiorespiratory response to a physiological stress such as hypoxia and more specific ally, may involve a severe bradycardia that transpires when a stress presents during sleep (77, 113, 149, 183) The relationship of SIDS to an infants sleep state is hypothesized to be associated wi th the immaturity of the sleep control system in infants and central adjustments in cardiorespiratory control that occur during the transition fro m quiet wake to sleep In humans, sleep is characterized by two main states: non-rapid eye movement (NREM) or deep sleep and rapid eye movement (234) or active sleep. NREM is typically defined by large amplitude slow wave electroencephalographic (EEG) recordings and low nuchal muscle tone. In adult mammals, NREM makes up approximately 80% of total sleep time (66, 67) In contrast, R EM is characterized by low amplitude high frequ ency EEG, even lower nuchal muscle tone and rapid twitch of the orbitals. Adult rodent sleep is characterized by similar states (NREM and REM), which take on similar properties including similar EEG and nuchal muscle patterns. Newborns, both human and rodents, however, do not demonstrate all adult -like sleep characteristics. In particular, as humans and rodents increase their postnatal ag e, there is a general incr ease in the length of time spent in wakefulness and decrease the time

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86 spent in REM (67, 107) Additionally, during early postnatal development both infants and rodent pups do not demonstrate typical adult EEG sleep patterns (66, 153) nor are circadianrelated sleep patterns present unt il the 3rd month of human development or postnatal day 16 (P16) in rodents (67, 68, 153) Infants at high risk for the SIDS do not demonstrate normal developmental sleep patterns, but rather have smaller developmental increases in the total time spent awake and spend more time in REM than infants with no family history or major risk factors for SIDS (34) Associated with these changes in sleep regulation, there is some evidence of sleep related changes in autonomic control, including abnormal changes in sympathetic and parasympathetic inputs to the heart during both NREM and REM sleep (119) Additionally, infants at high risk for SIDS demonstrated reduced arous al responses to hypoxia during sleep (48, 147) suggesting that SIDS may not only be related to changes in cardiorespiratory control during sleep but may also reflect a dysfunction of neural circuits involved in regulating the transitions from sleep to wakefulness I nterestingly, high risk female infants develop sleep patterning normally (33) a factor th ats likely to contribute to their almost 2-fold lower risk of SIDS (158) Changes in c ardiorespiratory function that typically occur in response to the transition from wakefulness to NREM in adults include a decrease in respiratory rat e (RR) and heart rate (HR) (124, 129) Respiratory changes also include an increase in breathing regularity in NREM sleep and HR changes include an increase in parasympathetic versus sympathetic drive (124) During REM sl eep, the respiratory pattern becomes irregular and there can be a general increa se in RR r elative to NREM sleep (129) HR in contrast, decreases in REM sleep relative to NREM sleep in adults

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87 (192) Interestingly, in infants, sleep state does not affect baseline HR until 3 5 months of age (251) an observation that suggests the first 6 months of human life are marked by complex integration processes within the brain between sleep and cardiorespiratory contr ol circuits. To our knowledge, no study to date has examined the effect of sleep state in rodent HR development, despite the fact that the rodent is a very common research model used to evaluate putative neurological changes that might contribute to SIDS, At present, one of the largest risk factors identified to contribute to SIDS is maternal smoking (157) and subsequentially, prenatal nicotine exposure (PNE) has bec ome a popular model for animal research investigating brain mechanisms underlying SIDS. Interestingly, PNE appears to accelerate the development of circadian rhythms in th e rodent (68) For example, PNE pups develop increased time s pent awake during the dark cycle compared to the light cycle and have an overall reduction in the time spent in REM sleep throughout early postnatal development than nonexposed pups. This consolidation of sleep may be associated with a deeper, m ore intens e sleep. Coincidentally, both SIDS victims and infants with maternal smoke exposure also demonstrate deeper sleep patterns (defined as reduced arousal re sponses) (33, 34, 193, 201) The current theories of SIDS causations have been furthered, most importantly, with the identification of specific developmental alterations in both cardiovascular and respiratory function. However, little is known about how PNE specifically alters the integration of the cardiorespiratory system to normal changes in sleep/wake state. Evidenc e from rodent brainstem slices suggests that PNE can indeed alter

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88 cardiorespiratory integration in cardiac vagal motoneurons (170) Unfortunately, how these PNE induced changes translate to effects on cardiovascular development during different sleep/wake states remain to be d efined. Thus, the present study was undertaken to evaluate changes in cardiorespiratory integration that occur during sleep versus wakefulness in both control (CON; salineexposed) and PN E pups beyond P10 when normal NREM sleep patterns begin to develop in the rodent. Additionally, since male infants are more likely to s uccumb to SIDS than their female counterparts (158) and few studies have examined the role of sex in both sleep state and cardiorespiratory development, both male and female offspring were evaluated. It was hypothesized that PNE individuals would exhibit a greater slowing of HR during sleep relative to quiet wakefulness (QW), particularly at P13 compared to CON offspring and these changes would be more severe in males Furthermore, it was hypothesized that any PNE induced differences during sleep would subsequently be abolished by P26 Methods All experiments were performed on Sprague Dawley rats (SD; Harlan Industries, Minneapolis, IN ) housed in the University of Florida (UF) animal care facility. All procedures were approved by the UF Institutional Animal Care and Use Committee (IACUC) prior to use. All rats wer e maintained under standard environmental conditions (12:12h light:d ark cycle) with free access to food and water. Rat dams (2304g) were implant ed with osmotic mini pumps (Durect Corporation Alzet Osmotic Pumps, Cupertino, CA) o n day 6 of gestation (of a normal 22 day gestation period). Dam s were briefly anesthetized to a surgical plane with isoflurane (5% initially, then 2 -3% for the remainder of the surgery) and a small incision made

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89 between the scapulae. A 28 day osmotic mini -pump (2ML4 Alzet) was inserted s ubcutaneously which allowed for 24 hour infusion of either nicotine tartrate (PNE, n=17: 6mg/kg/day of nicotine diluted in saline) or vehicle (CON, n=14: 2mL o f sal ine) (168) The nicotine dose was based on an estimated mid pregnancy weight gain of 54% gain from the initial weight taken at the time of pump implantation (average weight gain for a full term pregnancy was determined in preliminary studies of dams without pumps, n=8). Following subcutaneous positioning of the pu mp, the incision was closed and d am s were weaned from isoflourane (2.5% -0%) and allowed to awaken naturally from anesthesia Following a recover y period the dam s were returned to their individual cage s and observed daily until parturition. The day of par turition was termed postnatal day 0 (P0) On P 3, litters were culled to 8 pups to ensure proper nutrition for all pups. Pups were weaned on P21 and paired housed. All pups remain ed with the litter or littermates until the day they were selected for the stu dy, P 13, P 16 or P 2 6 Rat Pup I nstrumentation O n P12 14, or 24, pups were retrieved from their home cages and anesthetized to a surgical plane with isoflurane gas ( 5% in 100% O2 with a flow rate of 0.6 mL/min initially, then 2.5 3% for the remainder of the surgery) Following the induction of anesthesia, the h air on the nape of the neck was shaved and t he area was disinfect ed. A small incision was then made along the midline of the ventral surface of the neck. Two t hin wire paddled electrodes (PlasticsO ne Inc., Roanoke, VA) were inserted into the nuchal muscle bilaterally to record electomyogram activity (nEMG). T wo thin wire electrodes (Teflon coated; 0.005 mm diameter, A -M Systems Inc., Carlsborg, WA) were placed subcutaneously on both front limbs for electrocardiogram recording (ECG: Lead

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90 I). Another thin wire electrode was placed subcutaneously along the ventral surface of the back to serve as a ground wire The incision was closed with tissue glue (Nexaband S/C, Abbott Laboratories, Chicago, IL) and sutures. For P12 and P14 pups, all electrodes were connected to a back mounted pedestal (E363BM PlasticsOne Inc. ) that was secured in place with suture just on top of the shoulder blades. For P24 pups, four small holes were made into the skull positione d two at 2mm anterior and two at 2mm posterior to bregma with all four 3.5 medial lateral (100) In the anterior holes, two small electrodes were placed with small jewelers screws (PlasticsOne Inc. ). In the two posterior holes, two screws were placed to serve as a mount for the head stage. ECG and nEMG leads were placed as they were at P13 and 16. All electrodes and wires were secured into a head mounted pedestal (MS363 PlasticsOne Inc) that was secured to the skull with dental cement (Ortho-Jet, Lang Dental, Wheeling, IL) Following ins trumentation, all incisions were closed, p ups were weaned from isoflourane (2.5% 0%) and allowed to naturally awaken. A nimal were then allowed to recover on a warming blanket Post surgery all pups receive d subcutaneous fluids, analgesics (Buprenorphine, 0.01m g/kg), and topical antibiotic cream (Alpharma, Baltimore, MD). D uring recovery, the P12 and P14 pups were fed every half and hour with a milk substitute (30% fat milk based cream) Once able to right themselves the pups were returned to the mother and continually monitored until they beg an feeding on their own and the mother returned to grooming them Data Collection On the day of recording, pups were removed from their home cages Each pups pedestal was connected to a single brush 6 channel commutator (SL6C/SB PlasticsOne Inc. ) via a tether system allow ing for the minimal amount of movement

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91 restrictio n. The animal and tether was placed in a sealed plexiglass plethysmograph chamber (Buxco Electronic Inc, Wilmington, NC) The plethysmograph was fitt ed with a pressure transducer (TRD5700 Buxco Electronic Inc) to allow monitoring a respiratory related pressure changes and non-invasive recordings of respiratory rate (RR). Next the plethysmograph was placed in an electrically shield ed copper cage. A t hin wire thermister was inserted into one of the airflow holes in the plethysmograph and chamber temperature was continuously monitored. N ormal ambient temperature wa s maintained in the plethymograph wit h heat lamps to the temperature appropriate for each age (P1 3: 3334oC P16: 2930oC and P25:22oC) All experiments occurred between 1230 and 1700 h. For all age groups, output from the single brush commutator directed the ECG signal to Grass preamplifier w h ere the signal was a mplified and filtered. The ECG signal and the output from the plethysmograph pressure transducer were then sent through a real time data acquisition interface (Power1404, Cambridge Electronics Design (CED), Cambridge, UK) to a PC computer equipped with complimentary a data software system (Spike2, CED, Cambridge, UK). Data Analysis All data were analyzed off -line using Spike 2 software (Cambridge Electronic D e sign). For the duration of the recording, sleep wake state was scored in 10 s epochs (66) In P13 and P16, sleep wake state was quantified on nEMG activity and video monitoring (112) Those states include awake ( both A W : active wake which was high nEMG activity and visible movement and QW: quiet wake which was high nEMG activity with no visible movement ), NREM ( low nEMG activity and no visible movement or muscle twitching ) and REM (very low nEMG activity with presences of twitch). At P26

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92 sleepwake state was quantified similar to P13 and P16, but with the addition of EEG (W : high frequency -low amplitude EEG; NREM : low fr equency high amplitude EEG; REM : high frequency low amplitude EEG). For sleep time analysis, AW and QW were grouped together as wakefulness (W). For all physiological analysis just QW was used. HR was determined by the time interval between R wave peaks in the ECG leads. RR was determined by the time interval between pressure troughs, which represent the time of peak inspiration, in the plethysmograph tracing. HR and RR variability was analyzed with the nonlinear Poincare plots. From this analysis, two components were taken, SD1 and SD2. SD1 represents is the standard deviation that represents short term variability and the SD2 components represents long term variability (239) In HRV, SD1 is typically considered an estimate of parasympathetic tone and SD2 is an estimate of sympathetic tone (239) To determine significant developmental differences in pup weight, a threeway (postnatal day*sex*treatment) ANOVA was used. When indicated, a Scheffes post hoc analysis was used to compare groups. To determine significant developmental differences in HR and RR under specific sl eep/wake states, a two way ANOVA (postnatal day and treatment) was used to compare differences within the sexes To identify differences in how HR and RR change during sleep states, a oneway repeated measure ANOVA was used at each postnatal day within bot h sex groups. To examine all HR and RR variability components, a two way repeated measure ANOVA was used (behavioral state and treatment) at each postnatal day within each sex. When indicated, a Scheffes post hoc was run to identify significance across tr eatment groups and a Bonferroni adjusted paired student t -test was used to compare sleep states (P=0.016)

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93 P values of 0.05 or less were accepted as significant and all data are presented as meansSEM. R esults The average body weight of all animal included in data analysis are listed in Table 3 -1. Both sexes, regardl ess of treatment, significantly increased weight throughout the postnatal period (P<0.0001). Pup weight was significantly elevated at P26 compared to both P13 and 16. Weights tended to increase from P13 to P16 (P=0. 062). Sex also had a significant effect on pup weight (P=0.019). Post hoc analysis revealed that male pups at P26 weigh ed significantly more than females at the same age (P=0.001). PNE had no effect on animal weight at any of the days examined (Table 3 -1). Figure 3 1 depicts a typical recording from two control females one at P13 and the other at P26 during all three behavioral states which illustrates average developmental trends As identified in this figure, QW was characterized by high nEMG tone at both P13 and P26, and low amplitude, high frequency EEG activity in the P26 pups NREM was characterized by low nEMG tone and in P26 pups, high amplitude, low frequency EEG activity. Finally, REM was characterized by decrease in nEMG ton e relative to NREM except for periodic muscle twitching and in P26 low amplitude, high frequency EEG activity. With age, HR did not change noticeably, but there was a general slowing of RR independent of sleep wake state at P26 compared to P13. At P26 duri ng NREM pups appears to either not change RR compared to QW (males typically) or slightly increase it (females). Apparent changes in tidal volume between the two different ages is an art ifact of th e larger plethysmograph required for the larger animal.

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94 D evelopment of Sleep Patterning Figure 3 2 demonstrates th e average change in sleepwake behavior that occurred with maturation in male and female offspring. In male pups, the percent time spent in W tended to increase from P13 to P26 (P=0.076), but no PNE treatment effect was identified. NREM sleep time also tended to increase with age. Analysis of NREM sleep time identified a significant interaction between postnatal day and treatment (P=0.0028). Further analysis identified t hat P13 male PNE pups spent m ore time in NREM sleep than their CON counterparts (P=0.028). Additionally, only the CON males demons trated a significant increase in the percent ti me spent in NR EM from P13 and P16 t o P26 (P<0.0002). Finally, analysis of male REM sleep did not identify a treatment effect (P>0.05), but there was a significant effect of postnatal day (P=0.0001). Post hoc analysis demonstrated that the total time in REM was significantly greater in P13 pups compared to both P16 and P26 males (P<0.016). In female pups, like in males, no treatment or postnatal effect existed in the percent of total recording tim e spent in QW. N REM sleep times demonstrated only a significant effect of postnatal day (P=0.011). Although total time in NREM sleep tended to increase from P13 to P26, post -hoc analysis did not identify any specif ic significant differences (P>0.016). Finally, REM sleep time in the females also demonstrated a significant effect of postnatal day (P=0.006), but no treatment effect (P>0.05). Post hoc analysis demonstrated that time spent in REM at P26 in the females was significantly lower than at P16 (P=0.010). There was no significant difference between time spent in REM at P13 versus P16 or P26.

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95 Influence of SleepWake State on D evelopment of HR Figure 3 3 depicts the developmental changes in HR during each behavioral state in males (upper panels) and females (lower panels) of both treatment groups. No statistical difference between treatment groups across development was identified in males or females within any of the sleep wake states. However, visual inspection of the data identified that all male offspring tended to increase HR from P13 to P16 across all states. In cont rast, the changes in the female offspring across development appeared more gradual. Note however, that at P26, all pups demonstrated very little REM sleep during the one hour recording period. Therefore, only REM data from P13 and P16 are shown. To further evaluate HR changes as a function of sleep/wake state at each developmental age, HRs during sleep states (NREM and REM) were examined as a function of QW (Figure 3 4). At P13, male pups demonstrated a significant effect of treatm ent (P=0.05). Post hoc analysis demonstrated that the drop in HR from QW to sleep (NREM and REM) was significantly greater in the PNE males compared to the CON males (Figure 3 -4: upper left column; P=0.032). At P16, all males showed a decrease in HR during sleep compared to QW, but no treatment effect was identified (upper middle panel). Independent of treatment, however, at P16 the decrease in HR from QW was significantly greater during REM compared to NREM (P=0.004). Finally, at P26, since REM was not evaluated, only a treatment effect during NREM sleep was evaluated, but no significant effect was identified. In the female pups at P13 there was no treatment effect, but there was a strong trend for HR to drop more during REM compared to NREM from QW (Figure 4: low er left panel; P=0.058). Again at P16, there was no treatment effect for the females but

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96 with both groups combined, the drop HR during REM was significantly greater than the drop during NREM sleep (lower middle panel; P=0.0001). At P26, no treatment effect was identified in the change in HR during NREM (lower left panel). Influence of SleepWake State on D evelopmen t of R R Figure 3 5 describes the develo pmental changes in RR in each behavioral state. In male pups, no treatment effect in the development of RR during QW was identified (upper left panel). There was however, a significant effect of postnatal day (P<0.0001). Post -hoc analysis found that RR during QW at PND 26 was significantly lower than RR during QW at both P13 and P16 (P <0.0002). During NREM, a significant interaction of both treatment and postnatal day (upper middle panel; P=0.038) was identified. Pos t hoc analysis found that RR in the male CON pups at P16 was significantly elevated compared to RR in the PNE animals (P=0.017). Additionally, CON animals significantly dropped their RR at P26 compared to both P13 and P16 (P=0.002 for both). The PNE offspr ing however did not demonstrate any change in RR as postnatal day increased. No significant changes occurred in RR during REM as a function of either treatment of postnatal day in the males (upper right panel). Similar to the male offspring, RR of the female pups during QW demonstrated no treatment effects (Figure 3 -5; lower left panel), but did show a significant effect of postnatal day (P<0.0001). At P26, RR dropped significantly compared to RR at both P13 and P16 (P=0.0001 for all). During NREM, RR demonstrated a similar developmental pattern as QW and independent of any treatment effect demonstrated a significant effect of postnatal day (P<0.0001). At P26, in the females, RR during NREM was significantly lower than RRs during NREM at P13 and P16 (P<0.00 02). No developmental or treatment effects were identified in female REM RR.

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97 Figure 3 6 illustrates the average change in RR from QW during NREM and REM for both males (top panel) and females (lower panel). In the male pups at P13 no treatment effect was identified in how RR changed during sleep states (upper left panel). However, sleep state did significantly affect changes i n RR (P=0.001). D uring NREM RR decreased versus during REM, RR increased relative to QW, and the change in RR from NREM to REM was significant (P=0.0002). A similar relationship existed at P16; no treatment effect (upper middle panel), but REM RR was significantly elevated compared to the change in RR during NREM (P<0.0001). At P26, the change in RR relative to QW was small in the CON In contrast, in the PNE pups at P26 RR increased during the transition from QW to NREM sleep, and this was significantly different from the change observed in the CON group (upper right panel; P=0.013). Changes in RR from QW in the female offspring mimi cked the male pattern at P13 and P16. At both developmental time points there was no treatment effect and during NREM RR decreased relative to QW versus during REM, RR increased. For both P13 and P16 females, the increase in RR at REM was significantly dif ferent from the change in NREM (P<0.01). No differences existed between treatment groups in how females changed their RR from QW to NREM at P26. Sleeps I n fluence on HR and RR Variability To evaluate potential changes in autonomic and respiratory short -te rm and longterm variability, both HR and RR were examined using Poincare plots. A representative Poincare plot f or R -R interval from HR for two male pups one from each treatment group at P13 is shown in Figure 3 -7. SD1 represents short -term variability an d SD2 represents long-term variabilit y (239) In this e xample, the transitions from QW to NREM in the CON resulted in a decrease in both SD1 and SD2. In contras t, during

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98 REM sleep, relative to NREM, SD1 and SD2 increased and the pattern looked more similar to QW. In the PNE a similar pattern existed from QW to NREM, but the decrease in SD2 between states was not a great as observed in the CON animal. The transi tion from NREM to REM was marked by a small change in SD1 but little change in SD2 Male pup HRV The average change in HR short -t erm variability (HR SD1) and long -term variability (HR SD2) across sleep/wake state at specific developmental days for the mal e offspring are depicted in Figure 3 -8. Across all age groups there was a small decrease in HR SD1 during the transition from QW to NREM. This drop in HR SD1 however was not significant until P26 (upper right panel; P=0.00 2 ). The transition from NREM to REM was marked by a small increase in SD1 at P13 and P16, but this increase was not significant. No treatment effect was identified for HR SD1 at any age. Unlike SD1, state dependent changes in HR SD2 were more dramatic and a t P13 HR SD2 demonstrated a significant interaction of behavioral state and treatment ( Fig. 8, middle left panel; P=0.050). P ost -hoc analysis identified that PNE pups had a significantly lower SD2 component during QW (P=0.01 4 ) compared to CONs Additionally, while PNE pups demonstrated no state dependent changes, CON pups demonstrated a significant elevation in SD2 from NREM to REM (P=0.013). At P16 and P26 no treatment effect was found, but all male pups demonstrated a significant effect of state, including a significant reduction i n HR SD2 in NREM compared to QW at both P16 (P=0.001) and P26 (P=0.0001). In addition at P16 the rise in HR SD2 during REM was both significantly different from QW HR SD2 (P=0.002) and HR SD2 during NREM (P=0.0001).

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99 To assess any effect of PNE on autonom ic balance during sleep, the ratio of SD1 to SD2, or HR SD1/SD2, was also evaluated (Figure 3 8, lower panel). At P13, male pups HR SD1/SD2 demonstrated independent effects of treatment and behavioral state (P=0.027 and P=0.036 respectively). Post hoc anal ysis identified that HR SD1/SD2 in the male PNE offspring was significantly higher regardless of state (P=0.003). No state differences, however, were determined with post hoc analysis. By P16, male pups demonstrated no treatment effect, but behavioral stat e had become significant (P=0.0001). All male pups increased their HR SD1/SD2 from QW to NREM (P=0.008) and subsequently decreased their HR SD1/SD2 from NREM to REM (P=0.0001). Additionally, REM HR SD1/SD2 was significantly lower than QW. At P26, no treatm ent effect was identified, however, HR SD1/SD2 did significantly increase from QW to NREM (P=0.001). Female pup HRV All female pups changes in HR short term variability (HR SD1) and long term variability across sleep/wake state at specific developmental d ays are depicted in Figure 3 -9. Similar to males, the state dependent changes in HR SD1 were relatively small across all developmental time points. However, in the females a treatment effect was identified at both P13 for HR SD1. At P13, the PNE females demonstrated a significant elevation in SD1 regardless of behavioral state (upper left panel; P=0.015). By P16, the treatment effect was resolved in P NE females and all females demonstrated a significant state effect (upper middle panel). Post hoc analys is demonstrated that SD1 increased significantly from NREM to REM (P=0.002). No differences exist in SD1 at P26 regardless or treatment or behavioral state.

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100 State dependent changes in HR SD2 are exhibited i n the middle panels of Figure 3 -9. At P13 and P16 no treatment effects were identified but all females demonstrated a significant increase in HR SD2 during REM compared to both QW and NREM (P<0.003). At P26, the SD2 of female HR exhibited a significant interaction between of state and treatment (P=0. 023). Post hoc analysis however showed no significant treatment effect. Female CON animals at P26, however, did demonstrate a significantly lower HR SD2 during NREM compared to QW, an effect that was not identified in the PNE P26 females. Finally, analysis of H R SD1/SD2 in the female pups did not identify a significant treatment effect at any age (Figure 3 9, lower panel). How ever, at both P13 and P16 there was a significant effect of sleep/wake state and HR SD1/SD2 was significantly lower during REM compared t o both QW and NREM (P<0.005). At P26, HR SD1/SD2 increased significantly during NREM compared to QW (lower right panel; P=0.007). Male pup RRV Changes in the short -term variability (RR SD1) and longterm variability (RR SD2) of RR across sleep/wake state at specific developmental days were also evaluated (Figure 3 10 ). At P13 and P16 no significant treatment effect on RR SD1 was identified. However there was a significant effect of behavioral state (P<0.001) and at both P13 and P16 RR SD1 during NREM was s ignificantly smaller compared to both QW (P<0.002) and REM (P<0.002). In addition, at P13, RR SD1 during REM was significantly different from QW (P=0.018). At P26, behavioral state also had a significant effect on RR SD1 (upper right panel; P<0.0001) and NREM RR SD1 was significantly lower than QW. Additionally, male pups at P26 exhibited a significant treatment effect regardless of state (P=0.029), in that PNE male pups demonstrate lower RR SD1s.

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101 The lower panels in Figure 3 -10 demonstrate changes in RR S D2 across different sleep/wake states throughout development. At no developmental time point was a treatment effect identified. However, at all developmental time points RR SD2 demonstrated a significant effect of state (lower left panel; P<0.0001). The st ate dependent effect included a significant reduction in RR SD2 during NREM compared to both QW (P<0.0004) and REM (P=0.0001). Additionally, RR SD2 was significantly higher during REM compared to QW (P<0.003). Female pup R RV Short -term and long-term RR va riability for female pups at different ages are shown in Figure 3 11. Similar to the males at all developmental time points there was a significant effect with behavioral state ( P<0.0008), for both RR SD1 and RR SD2. This state dependent effect included a significant reduction in both RR SD1 and RR SD2 during NREM compared to QW (P<0.004). In addition at P13 and P16 there was a significant elevation in RR SD1 and RR SD2 during REM compared to QW and NREM (P<0.001). A significant effect of treatment was onl y identified at P16 for RR SD1. Post hoc analysis demonstrated that the RR SD1 for the PNE females was significantly lower than CON females regardless of state (P=0.013). D iscussion The present study was undertaken to evaluate the impa ct of PNE on cardio respiratory development during sleep. Postnatal times between P13 and P26 were evaluated since this is a critical time period for sleep consolidation and maturation of cardiorespiratory integration. Our results identified f our new findings. First, we conf irmed our original hypothesis that general differences occur in sleep time following PNE, including an increase in NREM sleep at P13 and this PNE induced change

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102 o ccurred exclusively in the male population. Second, we identified that the early maturation o f NREM sleep in the PNE males at P13 was associated with a significant lowering of HR from QW to NREM at P13, while both CON males and all female pups at the same developmental time point demonstrated a small increase in HR during NREM. Associated with this bradycardia during NREM in the PNE males was an overall elevation in the HR SD1/SD2 ratio during all behavioral states, indicating a possible abnormality in parasympathe tic t one. Third, although both PNE and CON females demonstrated similar state depend ent changes in RR when transitioning from QW to sleep, only female PNE pups at P16 demonstrated a significant reduction in RR SD1 (short term variability) that was evident across all behavioral states Finally, contrary to our original hypothesis that PNE induced differences would be abolished by P26, we did identify some subtle changes that persisted during sleep. In males this included an abn ormal reduction in NREM sleep time at P26 (PNE vs CON). This was coupled with a significant elevation in RR during NREM In females, P NE reduced the state dependent increase in H R SD2 or long term variability. These observations suggest that the effects of PNE on cardiorespiratory integration during NREM sleep may be permanent. SexB ased D ifferences in Sleep/ W ake D evelopment Although normal sex -based differences were not directly assessed in the present study, both CON males and females demonstrated a shift in the balance of time spent in REM versus NREM with maturation. These changes however appeared to be more gradu al in the female. For example, in females developmental changes in NREM sleep time were not significant. In contrast, males showed a more dramatic increase in NREM sleep time between P13 and P26 that was significant. Similarly, REM sleep time in males was significantly reduced between P13 and P26. Changes in REM sleep time in

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103 females were only significant between P16 and P26. These observations are in accordance with previous research that evaluated sex based differences in the development of sleep developm ent in piglets, which identified that female piglets develop indices of NREM sleep (particularly muscle atonia) more gradually than males (206) The more strikingly changes in sleep t ime in males may reflect some instability in central circadian maturation and may selectively predispose males to an inappropriate response to physiological challenge during sleep in early development. This may explain the greater incidences of SIDS in mal es (158) Impact of PNE on the Development of Sleep/Wake Time To our knowledge, only one other study has evaluated the impact of PNE on sleep/wake time in rodents. In that study, Frank and colleagues identified that PNE signi ficantly reduces overall sleep time between P12-P24 (68) This decrease in sleep time was mediated by a decrease in REM time compared to controls. In the present study however no significant change in REM sleep was identified. Alter natively, our results demonstrated that PNE induced a significant increase in NREM time specifically at P13 and this change was specific to males. The discrepancy between our results and Frank et al could be related to differences in recording protocols. W e examined only a one hour period of sleep/wake behavior versus Frank et al used a 24 hr recording protocol. Other studies examining normal developmental changes in sleep time using only a few hours of recordings have also demonstrated differences in devel opmental trends compared with studies that evaluated 24 hours (23) Alternatively, in the study by Frank and colleagues, male and f emale pups were not separated. Because our data suggest that male and female sleep times may be different throughout early

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104 development, not taking sex into consideration may change how the effect of PNE on sleep time is highlighted. Sexbased Differences on D evelopmental Changes in HR Regulation in Control Animals In the present study, HR differences between the sexes were not evaluated separately, primarily based on the lack of sex based differences in the previous chapter. However, the trend for HR to c hange during development appeared slightly different between the sexes. For example, males demonstrated an increase in mean HR from P13 to P16 across all three behavioral states (QW NREM, and REM). In contrast, female changes in HR were relatively steady between P13 -P26. This observation is similar to the developmental patterns we observed in sleep/wake time, in that female development may be more gradual versus males may undergo more dramatic shifts. A difference in the maturation of state dependent changes in HR was also identified between males and females. For example, at P13 state dependent decreases in HR during REM were more prominent in CON females compared to males. In contrast, this state dependent change in REM HR occurred at P16. The early ma turation of state associated changes in female HR was reflected in the Poincare analysis (SD1 and SD2), in that females demonstrated a significant effect of state on HR SD1/SD2 ratio at all developmental time points, but males did not demonstrate any state differences until P16. The mechanism underlying this sex based difference in maturation of state dependent changes in HR is currently unknown. T his suggest s th at female rodents may develop obvious state dependent changes in HR sooner than male offspring.

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105 Unlike HR, no obvious sex based differences in RR were identified. For example, both CON males and CON females demonstrated a drop in RR from QW to NREM and an increase in RR during REM at P13 and P16. These are changes that typical occur during the trans ition from wake to sleep in adults. Furthermore, developmental changes in RR during all states were similar between both CON males and females and there was a dramatic downshift in baseline RR at P26. A similar drop in RR by the third week of postnatal dev elopment has been reported by other investigators (96, 134) However, our results are the first to demonstrate that this developmental drop in RR occurs both in QW and NREM. This supports previous research which suggests that changes in maturation of central pattern generation for respiration are likely to be driving developmental changes in baseline RR. Indeed, significant changes in neurotransmitter sensitivity in brainstem regions critical to respiratory function, including the preBotzinger and NTS, have been demonstrated to occur throughout the devel opmental days examined (135, 136) Effect of PNE on D evelopmental C hange s in HR Control across B ehavioral States I n the present study, PNE animals did not demonstrate significant differences in mean H R throughout development within any of the behavioral states evaluated compared to CON pups However, when HR was examined during sleep as a function of QW, PNE induced a greater drop in HR selectively in males compared to CON. This PNE effect occurred only at P13 and did not occur at any developmental time point in females. This significant drop in HR in PNE males at P13 was associated with an elevated, although not signi ficant, HR during QW, a significant decrease in HR SD2 during QW and an elevation in the HR SD1/SD2 ratio regardless of state. Because the drop in SD2 during QW occurred in the absence of any change in SD1 and baseline HR

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106 was elevated, this raises the poss ibility that PNE may also have induced changes in intrinsic HR. Furthermore since parasympathetic drive may not be fully developed at P13, the primarily mechanism available to reflexively manage a possible increase in intrinsic HR would be through the symp athetic nervous system, expressed as a drop in SD2. Interestingly, i nfants at risk for SIDS and those exposed to maternal smoking also have been identified to have an elevated HR during QW (87, 205) and a significant slowing of HR during sleep (31, 85) Previous investigations u sing Poincare plots have demonstrated that during wakefulness SIDS infants present ed with lower overall beat to -beat variability compared to control infants (203) It is possible that changes in HR that occur during sleep in earl y development, when the cardiovascular regulatory systems are immature, contributes to a PNE infants increased susceptibility to SIDS. Moreover an exaggerated lowering of HR during sleep combined with a physiological stress, such as prolonged hypoxia, may induce an even lower HR (via recruitment of an immature parasympathetic system). The combination of these changes in autonomic control may challenge the immature cardiovascular control system beyond its ability to appropriately recover. Indeed, in P23 lambs PNE has been shown to cause an exaggerated bradycardic response to hypoxia that occurred specifically during sleep (83) It should be noted, however, that SIDS victims often present with an array of different HRV parameters (119, 200, 203) and only a subset of these infants demonstrate any particular change (203) Thus, the use of HRV as an indicator of SIDS risk may only be predictive of infants with changes in either autonomic function or intrinsic HR dysfunct ion. Moreover, autonomic blockad e studies have demonstrated that

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107 SD1 and SD2 are not pure estimates of parasympathetic and sympathetic drive (239) Thus conclusions based on time analysis of HRV should be used conservatively. Sexbased Differen ces the Effect of PNE on D evelopmental C hange s in HR Control across B ehavioral States In the present study the only significant differences identified in female HR development after PNE were found using HRV analysis. Similar to males the effect of PNE on HRV was significant at P13 and was not state selective. However, unlike males, the effect of PNE presented only in the HR SD1 an d not HR SD2 or HR SD1/SD2 ratio. If HR SD1 reflects parasympathetic drive, then this observation suggests that the effect of PNE in females was to increase baseline parasympathetic tone or accelerate the mature of parasympathetic integration. Indeed a put ative indication of premature maturation/integration of parasympathetic regulation was identified in the previous chapter by evaluation of the presence of RSA. Furthermore, if PNE has an effect on intrinsic HR, our results suggest there are sex based diffe rences in how the autonomic system compensates. These sex based differences in inherent autonomic development and compensatory mechanisms may be based in early brain sex based differentiation. At P26, both males and females demonstrated a drop in HR from QW to NREM and PNE attenuated this drop although not significantly in either sexes. However, HRV analysis identified a significant treatment effect of PNE in the females. Furthermore, this treatment was selective to HR SD2; PNE female did not decrease SD2 in NREM relative to QW, the typical pattern observed in all CON animals Since SD2 may reflect a change in sympathetic drive, the observation that PNE females did not decrease SD2 during NREM suggests that one persistent effect of PNE may be to alter basel ine sympathetic tone. Indeed, in the previous chapter we identified that PNE males respond

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108 to acute hypoxia with an abnormal and sustained tachycardia. Although the potential effect of PNE on the sympathetic drive (SD2) was less dramatic in females this supports other investigators observations that PNE effects are persistent (27, 217) and may induce more obvious differences in sympathetic tone in males (247) Effect of PNE on Control of RR during D ifferent B ehavioral State across Development s In t he previous study, RR was evaluated independent of state and t he primary effect of PNE on resting RR w as an elevation in males and a reduction in females compared to sex matched contr ol s In contrast, when RR was examined as a function of sleep state a significant reduction in RR was actually demonstrated during NREM at P16 in PNE males compared to controls. Conversely in the females when the animals state was taken into consideration there was a general trend for RR to be slightly higher in PNE females than CON females across all states at P13 and P16. All animals demonstrated an increase in RR during REM relative to QW, but there was no significant effect of PNE. It was noted however that the increase in RR during REM was slightly greater in the PNE animals (males and females). Because the observations from these two studies are somewhat contradictory in regard to the effect of PNE on RR, this highlights the importance of evaluating r espiratory function as a state dependent phenomenon. To further evaluate subtle differences in RR, RRV was used. The only significant effect of PNE on early postnatal development was identified in females as a reduction in RRV (RR SD1) independent of stat e. This is likely to be the result of an overall elevation of RR in the PNE female across state. In contrast, PNE males demonstrated this reduction in RRV only at P26. Although the origin of this reduction in short term

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109 variability is unknown, our results confirm reports of reduced RRV in SIDS infants (202) However, it was a little surprising that we only demonstrated this reduction in RRV in females. It is possible that a PNE related change in RRV in mal es occurred at either an earlier or later time point than was evaluated in the present study. Although, further study is needed to identify whether RRV changed significantly at earlier time points, our observations suggest that dramatic changes in RR are n ot present when an animal s sleep/wake state is taken into consideration. However if there is an effect of PNE on RR, it may be relatively subtle, but may persist in the males. Methodical Consideration s We felt it was important to maintain as much matern al care as possible during physiological testing, since maternal separation alone can induce many adverse effects on sleep and cardiovascular development (92, 221, 235) As a consequence our results confirmed previous studies which demonstrated PNEs ability to accelerate the development of sleep/wake function in rodents. In our study, the accelerated development was through an increase in NREM time, not a reduction in REM as previously reported (68) It is possib le then that by not examining a full 24 hr period, our results are only a small snapshot of sleep/wake behavior. The present work also examined rat pups shortly after they had been instrumented (the next day for P13 pups). It is possible that surgery stre ss was involved in the present study. Since we have previously shown an altered stress response (aim #1) and PNE is known to alter the hypothalamic -pituitary adrenal axis (18 4) it is possible the present results are related to stress only. However, the first two weeks of pain responses typically are related to increased sympathetic drive (103) whereas we demonstrated an increase in parasympathetic activity and/or a decrease in sympathetic drive.

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110 Additionally, surgery recovery time in kittens has not been shown to affect overall developmental and sleep r elated changes in autonomic function (51) Summary The present study provides an evaluation of the impact of sex on the changes a fter PNE in the integration of sleep/wake state on cardiorespiratory function during the second week of postnatal development. Our results suggest that sleep was the most vulnerable to physiological abnormalities after PNE. In general, males appeared more vulnerable to sleep dependent changes in HR, RR and central control of both systems than females after PNE. Sex differences in sleep/wake maturation may account for the gender bias currently present in SIDS mortalities. Moreover, because RR is very dependent on sleep/wake state, the effects of PNE on average RR were actually reversed when behavioral state was considered; suggesting that future evaluation of PNEs effect on respiration should always include identification of sleep/wake state. Interestingly although the majority of research into SIDS and PNE had focused on RR, our results suggest that the effects of PNE appear to be more dramatic for cardiovascular regulation. Our results confirmed previously reported alterations in parasympathetic function and subsequent cardiovascular abnormalities related to specific sleep states. Similar to the results presented in the previous chapter, we also identified that PNEs effects persist in a sex dependent fashion. In females, this was reflected by a change in HR SD2. In males, this was reflected by an inappropriate increase in RR during NREM and a reduction in RRV (SD1).

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111 Table 3 1. Average weight for all groups CON PNE Male Female Male Female P13 Weight (g) 281 282 292 261 N 6 5 5 6 P16 We ight (g) 322 301 342 322 N 8 7 6 7 P26 Weight (g) 693# 623*,# 691# 601*,# N 9 6 10 7 Data represented as meanSEM, indicates significant sex effect at P26 regardless of treatment, # indicates P26 is significantly different from P13 and P16 regardless of treatment.

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112 Figure 3 1 Representative tracing of all three behavioral states at two developmental ages (P13 and P26) in control females Left columns demonstrate quiet wake (QW), a state characterized by high nuchal EMG (nEMG) activity, mild variability in respiratory rate (RR) and in P26 a high frequency EEG. Middle columns demonstrate non-rapid eye movement (NREM), which is characterized by low nEMG activity, regular RR and in P26 a low frequency EEG. The right columns demonstrate rapid eye movement (REM) sleep, which is characterized by very low nEMG activity, highly variable RR and in at P26 high frequency EEG As illustrated i n the plesthymograph trace (PG), RR decreases from P13 to P26. However, apparent changes in tidal volume shown are an ar tifact of different sized testing chambers

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113 Figure 32. Developmental changes in the percent of total recording time spend in each behavioral state in both sexes. Males are represented in the upper panels by circles. Females are represented in the lower panels by squares. Wakefulness (W) is represented in the left columns. Non-rapid eye movement (NREM) is represented in the middle columns. Rapid eye movement (REM) is represented in the right columns. Control (CON) animals are represented by open shapes. Animals prenatally exposed to nicotine (PNE) are represented by closed shapes. + indicates a significant differences from CON. # indicates a significant difference from both P13 and P16 in the CON pups only. indicates significant differences between two states regardless o f treatment .

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114 Figure 3 3. Developmental changes in heart rate (HR) during three behavioral states. Males are represented in the upper pa nels by circles. Females are represented in the lower panels by squares. Quiet wake (QW) is represented in the left columns. Non-rapid eye movement (NREM) is represented in the middle columns. Rapid eye movement (REM) is represented in the right columns. H eart rate (HR) is represents as beats/min. Control (CON) animals are represented by open shapes. Animals prenatally exposed to nicotine (PNE) are represented by closed shapes.

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115 Figure 34. T he effect of sleep state (NREM and REM) on heart rate changes (HR) as a function of quiet wakefulness (QW) during three developmental time points. Males are represented in the upper panels. Femal es are presented in the lower panels. Postnatal day (P)13 is represented in the left columns. P16 is represented in the middle columns. P26 is represented in the right columns. Control (CON) animals are represented by open bars Animals prenatally exposed t o nicotine (PNE) are represented by closed bars. indicates a significant difference from NREM without treatment effect. + indicates a significant difference from CON animals. (NREM: non-rapid eye movement; REM: rapid eye movement).

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116 Figu re 35. Developmental changes in respiratory rate (RR) during three behavioral states. Males are represented in the upper panels by circles. Females are represented in the lower panels by squares. Quiet wake (QW) is represented in the left columns. Nonrapid eye movement (NREM) is represented in the middle columns. Rapid eye movement (REM) is represented in the right columns. Respiratory rate (RR) is repre sents as breaths/min. Control (CON) animals are represented by open shapes. Animals prenatally exposed to nicotine (PNE) are represented by closed shapes. + indicates a significant differences from CON animals at the postnatal day indicated. # indicates a significant difference from P13 and P16 in only CON animals. indicates significant differences between two states regardless of treatment.

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117 Figure 3 6. The effect of sleep state (NREM and REM) on respiratory rate changes (RR) as a function of quiet wakefulness (QW) during three developmental time points. Males are represented in the upper panels by circles. Females are presented in the lower panels by squares. Postnatal day (P)13 is represented in the left columns. P16 is represented in the middle columns. P26 is represented in the right columns. Control (CON) animals are represented by open bars. Animals prenatally exposed to nicoti ne (PNE) are represented by closed bars. NREM: non -rapid eye movement; REM: rapid eye movement. indicates a significant difference from NREM without treatment effect. + indicates a significant difference from CON

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118 Figure 37. Representative Poincare plots of heart rate intervals (RR) in two male pups at postnatal day 13.

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119 Figure 38. The effect of behavioral state on the short (SD1) and long term (SD2) heart rate variability in male pups. SD1 is represented in the upper panels. SD2 is represented in the lower panels. Postnatal day (P)13 is represented in the left columns. P16 is represented in the middle columns. P26 is represented in the right columns. Control (CON) animals are represented by open circles. Animals prenatally exposed to nicotine (PNE) are represented by closed circl es. QW: quiet wakefulness; NREM: non-rapid eye movement; REM: rapid eye movement. ^ indicates significant difference from NREM in CON animals only. + indicates significant differences between CON and PNE animals. indicates significant differences between two states regardless of treatment.

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120 Figure 3 9. The effect of behavioral state on the short (SD1) and long term (SD2) heart rate variability i n fe male pups. SD1 is represented in the upper panels. SD2 is represented in the lower panels. Postnatal day (P)13 is represented in the left columns. P16 is represented in the middle columns. P26 is represented in the right columns. Control (CON) animals are represented by open squares. Animals prenatally exposed to nicotine (PNE) are represented by closed squares. QW: quiet wakefulness; NREM: non-rapid eye movement; REM: rapid eye movement. + indicates significant differences between CON and PNE animals. & indicates significant difference from QW in CON ani mals only. indicates significant differences between two states regardless of treatment.

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121 Figure 310 The effect of behavioral state on the short (SD1) and long term (SD2) respiratory rate variability in male pups. SD1 is represented in the upper panels. SD2 is represented in the lower panels. Postnatal day (P)13 is represented in the left co lumns. P16 is represented in the middle columns. P26 is represented in the right columns. Control (CON) animals are represented by open circles. Animals prenatally exposed to nicotine (PNE) are represented by closed circles. QW: quiet wakefulness; NREM: no n -rapid eye movement; REM: rapid eye movement. + indicates significant difference from PNE animals with no behavioral state effect. indicates significant differences between two states regardless of treatment.

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122 Figure 311. The effe ct of behavioral state on the short (SD1) and long term (SD2) respiratory rate variability in fe male pups. SD1 is represented in the upper panels. SD2 is represented in the lower panels. Postnatal day (P)13 is represented in the left columns. P16 is represented in the middle columns. P26 is represented in the right columns. Control (CON) animals are represented by open squares. Animals prenatally exposed to nicotine (PNE) are represented by closed squares. QW: quiet wakefulness; NREM: non-rapid eye movement; REM: rapid eye movement. + indicates significant difference from PNE animals with no behavioral state effect. indicates significant differences between two states regardless of treatment.

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123 CHAPTER 4 IMPACT OF PRENATAL N ICOTINE ON THE HYPOT HALAMIC OREXIN SYSTEM: A POTENTIAL FOR EPIGEN ETIC CHANGES Introduction O rexin (also known as hypocretin) was first discovered in 1998 when the gene sequence was being investigated as an integral player in the control of feeding behavior (40, 193) However, following identification of the associated gene sequence, the neuropeptide has been more tightly linked to the regulation of arousal responses and the stability of sleep states The cell bodies of these neurons are exclus ively located within three anatomically adjacent regions of the hypothalamus, the lateral hypothalamus (LH) (244) the perfornical area (PeF) and the dor somedial hypothalamus (DMH) (180) but make widespread efferent projections throughout the brain. Each of the three areas identified to contain orexinergic neurons appear to play a slightly different role in various physiological functions. Cells located in the LH for example are often thought to be related to sleep/wake function while those in the DMH are related to stress and arousal state (88) Orexins role in the promotion of wakefulness was first demonstrated when a mutation in the gene for the receptor was linked to canine narcolepsy (133) Narcolepsy is characterized by excessive daytime sleepiness and the sudden onset of bouts of s leep that occur randomly and can last from seconds to minutes. Knockout (KO) mice missing either the gene for orexin (preproorexin) or the associated receptors (Type 1 or A and Type II or B receptors) have highly fragmented sleep with rapid transitions be tween states (111) Orexin KO mice also demonstrate devel opmental delays in the maturation of sleep patterns and have overall decreased arousal (22)

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124 Aside from promotion of wakefulness, orexin neurons also project to important cardiovascular control centers in both the hypothalamus and brainstem (58, 122) Intracerebroventricular administration of orexin causes both a tachycardia and pressor response. Subsequent administration of intravenous injection of an al pha(1) adrenergic or a betaadrenergic receptor antagonist attenuates this orexin-induced increase in arterial pressure and heart rate (7), suggesting that orexins ability to alter autonomic function is primarily via activation of the sympathetic system. Orexin can also alter respiratory function during specific behavioral states. For example, knock out mice demonstrate significant differences in the respiratory response to hypercapnia during wakefulness, but not during non-rapid eye movement (NREM ) sleep (125) Orexin neurons send their densest project of efferent fibers to the locus coeruleus (LC). The LC has been proposed as an integral player in central chemoreception (21, 182) Thus, it is possible than that orexin plays an important role in coordinating the respiratory and cardiovascular systems response to stressful physiological stimuli. Maturation of the brain orexin system parallels the developmental changes observed in previous chapters. For example, orexin neurons and axonal projections are present immediately after birth but the neuronal population appears to be relatively static until P 13. At this time point, orexin neurons undergo an extensive reorganization by increasing both in neuronal number and axonal projection density (225, 249) With similar developmental time courses, it is possible that maturation of the orexin system is also integrally involved with autonomic development, specifically as it relates to changes in blood pressure and heart rate during different sleep wake states or during arousal in response to physiological stress, as described in the previous two chapters. Animals

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125 exposed to nicotine prenatally (PNE) have been reported to demonstrate premature maturation of circadian rhythms and consolidation of sleep/wake bouts (68) raising the possibility that PNE may alter t he orexin neuronal development, and this central alteration may be an important contributing factor to the developmental changes associated with PNE described in earlier chapters and may play a role in sudden infant death syndrome. Yet, to date the impact of PNE on orexin neuronal function has not been evaluated. In the adult rat chronic nicotine exposure has been shown to cause an increase in prepro orexin mRNA expression within the hypothalamus and the expression of orexin peptides specifically in the D MH (110) I n a separate study, the up-regulation of orexin expression in response to chronic nicotine exposure was shown to be associated with decreased recep tor binding affinity in regions of the hypothalamus (109) Other areas e xamined in the basal forebrain however did not demonstrate this down-regulation of receptor sensitivity. These observations support a hypothesis that PNE could chronically alter an individuals orexin system Since orexin is involved in wake promotion and arousal response, alterations of the orexin system could explain the elevated wakefulness and altered stress responses observed in P NE pups. The present study then was undertaken to characterize both the transcriptional activity of preproorexin with the hypothalamus and the morphology of the orexin neurons within all three areas of the hypothalamus where orexin neuron cell bodies are found (LH, PeF and DMH) in age matched control and PNE offspring. Based on the physiological data presented in the previous chapters, we hypothesized than PNE would alter the level of prepro orexin mRNA within the hypothalamus in male animals

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126 only. To evaluate whether changes in mRNA expression were associated with changes cell morphology we also examined orexin cell size and number. It was hypothesized that PNE pups would demonstrate no change in the overall number or size of orexin positive cells within the hypothalamus but because stress responses have been demonstrated to be different in PNE pups the number of orexin neu rons populating the different subdivisions in the lateral hypothalamus (LH, PeF and DMH) would be altered compared to control animals. M ethods All experiments were performed on Sprague Dawley rats (SD; Harlan Industries, Minneapolis, IN ) housed in the Uni versity of Florida (UF) animal care facility. All procedures were approved by the UF Institutional Animal Care and Use Committee (IACUC) prior to use. All rats wer e maintained under standard environmental conditions (12:12h light:dark cycle) with free acce ss to food and water. PNE Exposure Rat dams (2407g) w ere implant ed with osmotic mini-pumps (Durect Corporation Alzet Osmotic Pumps, Cupertino, CA) o n day 6 of gestation (of a normal 22 day gestation period). Dam s were briefly anesthetized to a surgical p lane with isoflurane (5% initially, then 2 -3% for the remainder of the surgery) and a small incision made between the shoulders blades. A 28 day osmotic mini pump (2ML4 Alzet) was inserted subcutaneously which allow ed for 24 hour infusion of either nicot ine tartrate (PNE, n=9: 6mg/kg/day of nicotine diluted in saline) or vehicle ( CON, n=5: 2mL of saline) (168) The nicotine dose was based on an estimated mid -pregnancy weight gain of 54% gain from the initial weight taken at the time of pump implantation (average weight gain for a full term pregnancy was determined in preliminary studies of dams

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127 without pumps, n=8). Following subcutaneous positioning of the pump, the incision was closed and d am s were weaned from isoflourane (2.5% 0%) and allowed to awaken naturally from anesthesia Following a recover y period the dam s were returned to their individual cage s and observed daily until parturition. The day of parturition was termed postnatal day 0 (P0) On P 3, litters were culled to 8 pups to ensure proper nutrition for all pups. Pups were weaned on P21 and paired housed. All pups remain ed littermates until the day they were selected for the study, P242 6 This late day in postnatal development was chosen because orexin neuron developmental migration is complete and cell sizes are similar to those in the adult (225) Therefore, we hoped to minimize the individual variability associated with development. To avoid litter effects, no more than two pups were used from each litter. Tissue Coll ection Real timePCR ( RT PCR ). For RT -PCR analysis, a group of animals (CON males : n=5; CON females: n=5; PNE males: n=6; PNE females n=7) were heavily anesthetized with isoflurane and decapitated. The brain was removed from the skull and the hypothalamus w as isolated in an RNase-free environment Since orexin mRNA is made exclusively in the hypothalamus only a section of brain containing the hypothalamus was dissected The is olated tissue was cut in half lengthwise, placed in individual RNAse -free tubes a nd immediately frozen with liquid nitrogen. Each sample was then stored at 80C until processed. Immunohistochemistry. For immunohistochemistry, a separate group of animals (CON males: n=6 and PNE male: n=7) were overdose d with an intrapertonial injection of sodium pentobarbital (80-100 mg/kg; Sigma Chemicals, St. Louis, MO). To prevent blood clots during perfusion, a 0.2 mL injection of heparin (Henry Schein Inc,

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128 Melville, NY) was given into the left ventricle prior to perfusion. Animals were then immedi ately transcardially perfused with saline (0.9% NaCl) followed by 4% paraformaldehyde (Sigma). Brains were removed and soaked overnight in 4% paraformaldehyde and then placed in a 30% sucrose solution for 48 hours to ensure cryoprotection. The tissue was f rozen, coronally sectioned (40m) using a cytostat ( -20 C: Zeiss HM101, Carl Zeiss, Inc., Thornwood NY) and then placed in phosphate buffered solution (PBS) until processed for relevant protein. mRNA Isolation Previously frozen tissue samples (0.10.2 g) were placed in 1 mL of Trizol (Invitrogen, Carlsbad, CA). The samples were homogenized using a Polytron homogenizer (Tekmar, Janke and Kunkel, W Germany). Before samples were homogenized, the apparatus was cleaned by running it in sequential solutions of 10% bleach, 1% SDS, 1M NaOH, and two washes in HPLC water. Between each sample, the apparatus was cleaned by sequentially operating the homogenizer in 1% SDS and two washes of HPLC water. Samples were then centrifuged for 10 minutes at 12,000 x g at 4C. The supernatant was removed and transferred to a sterile tube. chloroform was added and samples were incubated for 5 minutes at room temperature. Samples were then centrifuged for 15 minutes at 12,000 x g at 4C. The upper aqueous phase was then removed and transferred to another sterile was added and samples again incubated for 10 minutes at room temperature, after which they were again centrifuged at 12,000 x g at 4C. The isopropanol was removed from each sample and t he remaining pellet was washed with 1 mL of 75% ethanol and centrifuged at 7500 x g at 4C for 5 minutes. The ethanol wash was removed and the remaining pellet was air dried until it was just translucent (1530 minutes). Each pellet

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129 was re-suspended in HPL made and all samples were stored at 80C. The absorbance of each sample was read by spectrophotometer (Nanodrop, Thermo Scientific, Wilmington, DE) to determine RNA concentration and purity. RT -PCR mRNA was quantified usi ng a twowere used to create cDNA with a high capacity cDNA archive kit (Applied Biosystems, to in a PCR reaction to identify orexin mRNA. All RT -PCR reac tions were run in triplicate in 96well optical grade plates in a Prism 7000 Sequence Detection System (Applied Biosystems). Orexin expression was determined by two -step quantitative RT -PCR using Syber Green (Syber Green Master Mix, Applied Biosystems). Se quences of primers for preproorexin were as follows: forward primer 5 -CATCCTCACTCTGGGAAA G -3 and reverse primer 5 AGGGATATGGCTCTAGCT C -3. Expression of 18s mRNA was determined by quantitative RT -PCR using Taqman probes and primers (Taqman one-step R T -PCR master mix, Applied Biosystems). Control reactions containing no template were run for each plate. Thresholds (Ct) were set in the exponential phase of amplification. Relative t (from triplicate) of the gene of interest (preproorexin) and the corresponding ribosomal RNA was subtracted from all other groups (CON females, PNE males and PNE females) t o

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130 expression relative to the CON group was calculated by using the formula 2 The percent change from CON males and standard error of each group was then calculated f rom each group. Immunohistochemistry Tissue sections from the hypothalamus were processed for or exin -A positive neurons. Only male pups were used for the orexin morphological measurements because our previously presented work identified that male pups wer e altered the most by PNE. Sections from each group were processed in parallel to ensure similar processing conditions. Briefly, the protocol was as follows. Free-floating sections were washed in PBS for at least 12 hours before processing. S ections were t hen rinsed in 3% goat serum (GS) and 0.4% TritonX (T) in PBS for one hour. After which, sections were incubated overnight at room temperature in rabbit anti orexin-A (1:10,000 dilution in 1% GS -T -PBS; Peninsula Laboratories, Inc., San Carlos, CA). The ne xt day, sections were rinsed in 1% GS -T -PBS for one hour and then incubated in goat anti rabbit antibody (1:500 diluted in 1% GS -T -PBS; Jackson Immunoresearch) for two hours. Once again, the sections were rinsed in 1% GS -T -PBS for an hour and then placed f or 30 mins in avidin biotinylated-peroxidase solution (ABC Vectastain Kit, Vector Labs). Following 30 mins, tissue was rinsed in 1% GS -T -PBS. The final visualization of orexin positive neurons was with a chromagen solution (0.05% diaminobenzidine, DAB: 2.5% ammonium sulfate; 0.033% hydrogen peroxide in 0.05 M T -HCL; Sigma). Slices were rinsed three times with PBS, mounted on glass slides, allowed to dry overnight and coverslipped (Vectamount, Vector Labs, Burlingame, CA).

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131 Microscope A nalysis of I mmunohistr ochemical L abeling Mounted tissue sections was imagined using a light microscope (Zeiss Axioskop, Thornwood, NY) equipped with a digital camera (Nikon) and color coupler (CPi) which were connected to a PC computer. Image capturing software, ImagePro ( Media Cybernetics, Besthesda, MD ) was used to ph otograph each sample. C ellular quantification software (Metamorph 6.3, Molecular Devices, Downingtown, PA) was used to analyze tissue sections for both neuronal counts and size. For each animal, two representativ e images were taken. The region of the hypothalamus selected for analysis included a region previously identified to be heavily interconnected with important brainstem autonomic centers (253) Figure 4 -2 illustrates the spec ific area of the hypothalamus selected for morphological analysis of the orexinergic neurons (253) As shown in Figure 4 2B, the region containing orexin-A positive neurons was subdivided into 3 subregions for analysis, incl uding the LH, PeF and DMH. Previous literature has suggested that the physiological function of these three subdivisions may be distinctly different, and as such they were quantified separately (88 ). As illustrated by the arrows in Figure 4 -2C orexin positive cells contained a clearly defined empty nucleus surrounded by a dark brown coloration in the cytoplasm Information regarding cellular morphology (cell size and numbers) was averaged to yield a representative count for each animal. The mean and standard error of these morphological measurements for each group was then calculated. Physiological Measurements In a separate cohort of male animals (CON: n=8,PNE: n=10), pups were retrieved from the ir home cages on P24 and anesthetized to a surgical plane with isoflurane gas and instrumented with bilaterally nuchal muscle EMG electrodes to record neck muscle

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132 activity (nEMG) and EEG electrodes were implanted in the skull as described in the previous c hapter to help stage sleep wake state. Following instrumentation, all incisions were closed, pups were weaned from isoflourane (2.5% -0%) and allowed to naturally awaken. Animal were then allowed to recover on a warming blanket Post surgery all pups rec eive d subcutaneous fluids, analgesics (Buprenorphine, 0.01m g/kg), and topical antibiotic cream (Alpharma, Baltimore, MD). Physiological Data Collection On the day of recording, pups were removed from their home cages Each pups pedestal was connected to a single brush 6 channel commutator (SL6C/SB PlasticsOne Inc. ) via a tether system allow ing for the minimal amount of movement restrictio n. The animal and tether was placed in a sealed plethysmograph (Buxco Electronic Inc, Wilmington, NC) The plethysmograph was fitted with a pressure transducer to allow monitoring a respiratory related pressure changes and noninvasive recordings apneas. Next the container was placed in an electrically shield ed copper cage. All experiments occurred between 1230 and 1700 h. Output from the single brush commutator directed the nEMG and EEG signal to Grass pre amplifier w h ere the signal was a mplified and filtered. The signal s and output from the plethysmograph pressure transducer were then sent through a real time data ac quisition interface (Power1404, Cambridge Electronics Design (CED), Cambridge, UK) to a PC computer equipped with complimentary a data software system (Spike2, CED, Cambridge, UK). Data Analysis All physiological data were analyzed off -line using Spike 2 software (Cambridge Electronic D e sign). For the duration of the recording, sleepwake state was scored in 10

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133 s epochs (66) Sleep wake state was quantified on nEMG and EEG activity and video monitoring. T hose states included wake (W : high nEMG activity high frequency -low amplitude EEG and visible movement ), non-rapid eye movement (NREM : low nEMG activity low frequency high amplitude and no visible movement or muscle twitching) and rapid eye movement (REM : very low nEMG activity high frequency low amplitude and the presences of muscle twitch). Total time spent in each state was then added together and divided by the number of times that state occurred to establish the average duration of each sleep/wake st age. To determine apnea occurrence index, the number of apneas in the one hour recording was counted. Because apneas primarily occur during either REM sleep or the transition from REM or NREM and/or sleep to wakefulness, the numbers of apneas in each stat e were analyzed separately. A two way ANOVA (treatment and sex) was used to calculate PNEs effect on orexin mRNA expression. Although mRNA expression data is presented as a percent change from CON males, t actually used to determine statistic ally significance. A oneway (treatment) repeated measure ANOVA was used to determine PNEs effect on the morphological parameters of orexin neurons. A two way ANOVA (treatment and sex) was used to calculate PNEs effect on behavioral state bout length. A one way (treatment) ANOVA was used to determine significance in apnea occurrences. When appropriate a Scheffes post hoc analysis was run to determine significance between treatments using a significant pvalue of 0.05. A Bonferroni adjusted paired t -test was used to determine significance between sleep states with a significant pvalue of 0.017.

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134 R esults RT -PCR Figure 4 1 illustrates the mean percent change in preproorexin mRNA levels within the hypothalamus as a function of the average expression in the CON males. A two way ANOVA identified both a significant effect of sex (P=0.001) and treatment (P=0.0 13). After post -hoc analysis, all PNE groups combined demonstrated significantly less preproorexin mRNA compared to the CON pups (P=0.05). Additionally, w hen all data from each sex was combined, females demonstrated significantly higher prepro orexin mRNA levels compared to males (P=0.0009). Orexin Cell Morphology Figure 4 3A is a representative picture from the DMH from each treatment group. As illustrat ed by this example, the PNE animals generally appeared to a slightly greater number of neurons with larger cell size. Although not quantified, the change in cell size and number was offset by what appeared to be a pruning or reduction in orexin containi ng axons or dendritic branches in the PNE animals. Figure 4 -3B and C illustrate the average number of orexin positive neurons and the average cell size of these neurons quantified for each subdivision of the hypothalamus. PNE had no significant effect on either cell number (Fig. 4 -3B) or cell size (Fig. 4 3C) in any of the hypothalamic areas examined. There was however a small reduction in the number of average number of neurons in the PeF region and a small increase in the number of neurons in the DMH. Orexin Physiology To evaluate whether the subtle changes in orexin expression induced by PNE exposure described above were associated with an alteration in orexin regulated

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135 behaviors, both sleep/wake state bout duration and number apneas during sleep were quantified from P26 males. As shown in Fig. 4 4A, bout duration demonstrated a significant effect of state (P<0.0001) and a significant interaction of both state and treatment (P=0.0093) During W, PNE animals tended to have significantly longer bout durations compared to the CON group (P=0.074 ). No differences were found in NREM bout duration. In contrast, during REM PNE animals demonstrated significantly shorter bout durations (P=0.014). Ad ditionally, PNE animals demonstrated significant changes in bout length across all three behavioral states. W was significantly longer than both NREM and REM (P=0.001 and 0.003 respectively) and NREM bout length is significantly longer than REM bout length (P=0.003) in the PNE animals. CON animals did not demonstrate t he reduction in bout length during NREM compared to REM. They did demonstrate a significant reduction from W to NREM (P=.0001) and REM (P=0.0004). Figure 4 4B illustrates the average number of apneas identified during REM sleep and the transition from sleep to wakefulness. PNE did not significantly change the number of apneas in either state (P= 0.372 and 0.267). Visually the PNE group appeared to have a reduction in the average number of apneas during state transition compared to the CON group; however thi s was simply an artifact of one CON animal that experienced an abnormally large number of apneas during state transitions. Discussion The present study investigated the potential effect of PNE on the modulation of the orexin expression in the hypothalamus. The results of this study confirm previous reports that orexin mRNA in females is, in general, significantly elevated compared to males (105) Although this sex -based difference persisted following PNE, our results

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136 demonstrated for the first time that PNE significantly reduce s orexin mRNA levels in the hypothalamus at P26 independent of sex based differences. This observation is unlike the effect of chronic nicotine exposure on prepro orexin mRNA expression in the hypothalamus of the adult rat (110) Interestingly, the changes in mRNA following PNE exposure were not associated with any significant change in overall cellular morphology or sleep wake behavior. This raises the possibility that changes in orexin expression may be more tightly linked to stress related behaviors since orexin has been shown to be integrally involved in defense or arousal responses (19) SexB ased D ifferences in Prepro -orexin mRNA Previous research has indicated that adult female rats have more prepro orexin mRNA in the hypothalamus compared to males (105) Additionally, when females are ovariectomized, orexin receptor type 1 mRNA increased in the pituitary and with estrogen treatment, receptor mRNA returns to baseline (104) These data suggest a strong link between estrogen and the orexin system In the present study we identified approximately a threefold increase in prepro orexin mRNA in P25 females compared to mal es. However, during the postnatal period examined in the present study the levels of sex steroids should be relatively low (47) Therefore, estrogen levels probably had no role or a minimal one on this sex based difference. Alternatively intrinsic sex differences in orexin activity have been demonstrated in fasting animals, where orexin neural activity was higher in females regardless of estrous cycle compared to males (69) T he sex based differences identified in the present study for prepro orexin mRNA is probably related to t he sexual differentiation of the brain, which occurred prior to the time period examined during the present study S exual differentiation of the brain is a product of varying estrogen levels within the first week of life when a male brain is

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137 actually differentiated by a large surge of testosterone that is then aromatized into estrogen (252) In effect, a large dose of estrogen is required during the early postnatal period to masculinize the brain. Therefore, orexin expression may have been permanently altered in a sexually dimorphic manner by P13 when the neurons begin to undergo a dramatic reorganization (225) If treatment wi th estrogen in OVX females causes a decrease in orexin responsiveness (decrease in orexin receptor) then it is possible that the postnatal surge of testosterone (converted to estrogen) in the male triggers this sex dependent downregulation of the orexin s ystem as well. It should be noted that previous work has demonstrated that only orexin receptors expression cha nged after sex steroid treatment; sex steroid appli cation itself did not alter preproor exin mRNA in the hypothalamus of the adult rat although orexin expression was higher in females compared to males (104) However, that does not exclude sex hormones from acting during perinatal development. Therefore, further studies should be performed at earlier time points to elucidate the cause of this sexual dimorphism. PNE I nduced C hanges in P repro orexin mRNA Expression In the present study PNE weanlings demonstrated significantly reduced mRNA expression in the hypothalamus. Indeed, at P26, PNE induced approximately a 50% reduction in prepro orexin mRNA levels in both male and female offspring. This outcome is unlike previous reports on the effect of chronic nicotine administration in adult which demonstrated an increase in orexin transcription within the hypothalamus (110) This suggests that nicotine exposure in utero acts through a different pathway tha n chronic exposure in the adult. Although the pathway mediating nicotines effect on orexin expression is currently unknown, there is some evidence for the involvement of relay neurons in NTS (255) that mediate both acute and chronic changes in other

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138 regions of the hypothalamus in response to nicotine administration (110) The effect of PNE on these pathways is unknown, but should be examined in future studies. Chronic nicotine also down-regulates the binding sites of orexin receptors within select brain regions (109) It is possible that PNE can differentially change aspects of the orexin sys tem, similar to chronic nicotine, even if it is modulating the system in different ways. However it is interesting to note that the effect of PNE on orexin expression did not involve the direct effect of nicotine since these animals were not exposed to ni cotine after birth. Thus, the changes in orexin cellular development that we have identified to occur postnatally must reflect the epigenetic eff ects of nicotine exposure during development. After PNE, orexin mRNA levels in the female offspring approached the levels of CON males. This may confirm our previous hypothesis that PNE acts as a masculizing event. The present data identifies a specific brain region that might be influenced in this manner. However, as mentioned above in the adult rat sex steroids have not been reported to specifically alter orexin mRNA levels. Further study is needed to identify if PNE is indeed acting on sex steroid production in utero to produce these postnatal changes and if it is this sex steroid dependent pathway that acts only during the perinatal period to alter orexin transcription. Neurotransmission however in general is a product of more than just mRNA and cellular morphology. Future studies should identify whether PNE also alters either orexin receptor transcription and/or receptor sensitivity. Additionally, orexinergic neurons project to a number of autonomic control centers, including its densest projection site, the locus coeruleus. The changes in the orexin system, particularly

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139 receptor function, after PNE must be exam ined in these areas as well. Additionally, much of the work presented in the previous chapters, suggest that cardiorespiratory abnormalities have a specific critical window during development. Therefore, orexin may demonstrate more severe abnormalities at a n earlier postnatal time point perhaps between P10 -P16. A more detailed study of PNEs effects on orexin neurotransmission and development will provide new insights into the potential role of this important neurotransmitter in mediating some of the abnor malities observed in offspring predisposed to SIDS. PNE Effects on O rexin C ell M orphology Since no changes were identified in the cellular morphology of orexin cells nor did orexin cells migrate to the DMH, we rejected our initial hypothesis regarding orexin morphology and migration. This lack of differences was surprising because orexin cells within the DMH are a proposed component in the stress response (88) The DMH itself is critical in produc tion of the fight or flight response (45) The significantly elevated HR response to hypoxia seen in PNE pups at P 26 suggested that these animals may be over activating this brain arousal center. With the DMHs, and specifically the orexin neurons within the DMH, known role in producing physiologically arousal, particular the tachycardia component, it is peculiar that we did not demonstrate a change in th ese cells. However, we only cho se one time point. It is possible that orexin demonstrates more obvious deficiencies during another developmental time point. Additionally, changes in orexin signaling may be more related to alterations in orexin receptor expression or sensitivity. The studies done in the chronic nicotine model do indeed suggest that orexin signaling can differentially change depending on specific brain regions. We suggest that orexin receptors in downstream arousal centers, such as the

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140 L C, or upsteam centers, such as the prefrontal cortex, may be more sensitive following PNE. PNE Effects on S leep wake B ehavior and A pnea I ndex Since much work on orexins physiologically activity has been in KO mice, we par alleled our data by examin ing two typical characteristics of these mice, decreased behavioral state bout length (22) and increased apnea number (254) in our rat cohort. In the present study, changes s imilar to orexin KOs were not identified in either of these physiological measurements. This result might have been expected given the different physiology of a complete KO versus an animal with simply less mRNA. Therefore one might expect more subtle abnormalities than a KO mouse. Indeed, in the present study we did demonstrate a significant alteration in behavioral state bout length after PNE. This result confirms other reports that PNE alters the sleep patterns, predominately by a reduction in REM sleep This decrease in REM (and subsequent elevation in W) may be a form of relative REM sleep deprivation. REM sleep deprivation is associated with a number of other disease states, including elevated risk of hypertension (152) and anxiety (228) and loss of normal memory function (131) It is possible that if PNE animals conti nue to demonstrate these abnormal sleep patterns they could be at increased risk for certain diseases later in life. Summary This study examined the potential effect of PNE on the hypothalamic orexin containing cells. We confirmed previous work that femal es in general demonstrate higher levels of orexin mRNA than males. However, our results confirm these sex differences at a developmental age with little sex hormone present. Like work done with chronic nicotine in adult rats, we demonstrated that PNE decreased orexin mRNA in the

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141 hypothalamus. The effect of PNE on orexin was independent of sex, but did appear to reduce orexin mRNA levels in the female to levels more similar to those observed in the age matched CON males. Taken together, our results suggest t hat changes in the orexin system may play a prominent role in the abnormalities in the development of sleep dependent changes in cardiovascular function which are associated with PNE. Future studies then should include an evaluation of both orexin projecti ng areas and receptor sensitivity to further determine the impact of PNE on the orexin system.

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142 Figure 41. Mean percent change in preproo rexin m RNA expression in the hypothalamus of 26 day old rat pups following prenatal nicotine exposure (PNE). All data are represented as a function of the average mRNA levels in the control (CON) males. Male animals (CON male: n=5 and PNE male: n=6) are represented by open bars. Female animals (CON female: n=7 and PNE female: n=5) are represented by the closed bars. represents significant (P<0.05) treatment effect; + represents significant sex effect.

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143 Figure 42. Representative image of the hypothalamic areas examined from a male animal. a tracing from Paxinos and Watson illustrating the area chosen for immunhistochemical examination which highlight s the location of the optic track (opt); B) an image outlining the three hypothalamic areas examined, which include the lateral (LH) perfornical (PeF), and the dorso-medial hypothalamus (DMH); C) higher magnification of image in B to highlight the cells c ounted must display a dark cellular body with an empty nucleus (f: fornix, mt: mammothalamic tract, hippo: hippocampus, VMH: ventro medial hypothalamus)

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144 Figure 43. Mean morphological measures of all male animals acro ss all three hypothalamic areas. A) Representative pictures from the DMH region in CON versus PNE. B) the mean cell count of orexin-A positive neurons within the lateral (LH), perfornical (PeF) and dorso-medial (DMH) hypothalamus and C) represents the mean cell size of orexin-A positive neurons in all three hypothalamic areas. CON animals (n=6) are represented by open bars. PNE animals (n=7) are represented by the closed bars. No significant differences existed in morphological measurements

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145 Fi gure 44. Physiological data from b oth PNE and CON male weanling rats. A). Bout duration of all three behavioral states examined, wake (W) non-rapid eye movement (NREM) and rapid eye movement (REM) States are divided by the two lines. Females are represented by closed bars. Males are represented by open bars. CON animals are on the left of side of section. PNE animals are grouped together on the right. No si gnificant differences existed. B) The occurrence of apneas in an hour recording during either REM or a transition between a state. Sex was not examined. CON animals (n=8) are represented by open bars. PNE animals (n=10) are represented b y closed bars. No significant differences were found.

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146 CHAPTER 5 SUMMARIES AND CONCLUSIONS Recent wor k to identify fundamental abnormalities associate d with SIDS has led to its classifi cation as a multi -factorial syndrome (121) Using epi demiological studies, researchers have developed a framework of risk factors for SIDS an d currently it is well accepted that maternal smoking substantially increase s that risk (155, 157) Since the n icotine in cigarette smoke has been implicated as an important culprit in this increased risk (216) animals models have used PNE in an attempt to mimic SIDS. Unf ortunately, PNE in animals is not an exact model, primarily because there is little evidence indicating that animals die suddenly during early postnatal development. Moreover, the liter ature on PNEs effect on respiratory maturation is conflicting (10, 96, 143) and work in infants suggests that SIDS may be more related to cardiovascular abnormaliti es (77, 113, 149, 183) T h e amount of research completed to date regarding th e impact of PNE on cardiovascular regulation has been limited. Ther e is evidence however that PNE rodents have significantly decelerated HR responses to hypoxia (219) and in vitro brain stem slices have demonstrated that PNE creates abnormalities in GABA activity in the cardiac va gal neurons (171) This limited amoun t of cardiovascular -related research that has been done on PNE rodents has focused mainly on rodents of very young postnatal ages (P1 P10). However, it has been suggested that in the rat this early time period actually correlates with the third trimester o f human development (186) Indeed, rodent sleep/wake maturation does not reorganize until the end of the second postnatal week (68) and the parasympathetic system does not play an important role of resting HR until P16 (114, 242) Therefore, a n examination of cardiovascular regulation using later postnatal time points in the rodent may be more relevant t o uncovering potential

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147 abnormalities associated with PNE and SIDS. Moreover, since epidemiological evidence has also identified that the incidence of SIDS is greater in males, factoring sex -based differences associated with PNE, especially in relation to i ts cardiovascular effects, is also important. In the studies presented above, we examined the potential for PNE to induce sex dependent changes in cardiorespiratory control between the second and third week of life. We hypothesized that PNE would accelerat e the developmental integration of sleep/wake cycles and cardiorespiratory function, but that these abnormalities would be most prominent in the m ale. The following is a summary of the findings from the experiments. Summary of Findings Aim #1: Impact of PN E on the A cc eleration of Cardiorespiratory Function Aim #1 sought to characterize the sex dependent changes in cardiorespiratory integration and the hypoxic response after PNE. Based on previous literature it was hypothesized that PNE would cause a premature integration of the cardiovascular and respirator y systems in male offspring only, such that PNE male pups would demonstrate an accelerated maturation of RSA and a blunted HR response to hypoxia. In accordance with our hypothesis, the results of aim #1 demonstrated that, under resting conditions, PNE did induce accelerated integration of respiratory input on parasympathetic modulation of HR. Interestingly, this accelerated development was exemplified by a possible overshoot in the correlation or slope of the relationship bet ween HF peak location and RR, a pattern which CON animals never demonstrated. Furthermore, although not predicted, female PNE pups also demonstrated this overshoot pattern of RSA integration. In PNE females elevated RSA occurred at P13 and was resolved by P1 6. In contrast, PNE males expressed this RSA overshoot at

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148 P16 and it was resolved by P26. These observations suggest that the presence of an overshoot in RSA may be indicative of central cardiorespiratory control abnorm alities. Interestingly the PNE induced changes in RSA occurred in both sexe s, but was expressed at an early time point in females. Because this RSA abnormality was resolved by P16 in females but became evident in males by P16 suggests that s ome of the s ex -dependent effects of PNE on SIDS may be related to inherent differences in brain maturation between males and females. I f males present with an abnormal pattern of cardiorespiratory integration (RSA overshoot) at the same developmental time point tha t other brain control circuits are also undergoing rapid reorganization, their ability to respond to physiological stresses may be severely compromised. In response to an acute hypoxic exposure PNE did not induce any significant changes in RR in eithe r sex. Rather, PNEs impact on the hypoxic response appeared to be limited to changes in HR and in this instance was male -specific. For example, at P13 the HR response to hypoxia was significantly blunted in PNE males compared to CON males. In addition at P16, although no t significant from CON males, the PNE males appeared to have a delayed HR response to hypoxia. This observation combined with evidence of developmental abnormalities in RSA suggest s that the impact of PNE in males has a greater effect o n cardiorespiratory function during early postnatal development compared to females and may explain why males dem onstrate higher SIDS rates Finally, the results of aim #1 also identified longer lasting effects of PNE than were originally hypothesized At P26, male PNE pups demonstrated significantly elevated HRs during hypoxia compared to CON males. This was a sex -dependent change and

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149 did not occur in females. This finding suggests that PNE may cause enduring changes in central autonomic regulation whic h persist in males and may increase an individuals risk for cardiovascular disease later in life. Aim #2: Impact of PNE on the Acceleration of Behavioral States Influence on Cardiovascular Function Aim #2 sought to characterize PNEs effect on sleep/wak e state integ ration with cardiore spiratory function. Although no work to date has examined the effect of PNE on sl eep dependent changes in the rodent cardiovascular system PNE has been shown to accelerate the development of sleep/wake systems alone (68) and SIDS is often associated w ith alterations in sleep /wake function (34) Therefore, we proposed to examine PNEs effect on the development of state dependent changes in HR and RR. We also included sex as a possible contributing factor in these changes for the same reasons presented in the previous aim. It was hypoth esized that PNE would induce a sex -dependent acceleration of the integration of sleep/wake state on HR function. The results of aim #2 were in agr eement with previous reports which suggest ed that the normal time course for female postnatal development of state-dependent changes in cardiorespiratory function are more gradual than in male s. The m ore abrupt or dramatic developmental changes observed in males suggest that males may undergo more periods of neural network instability or dramat ic reorganization. This apparent sex -dependent difference may make it difficult for males to compensate for intrinsic abnormalities or extrinsic insults at specific developmental time periods. Additionally, the results of aim #2 reinforced previous work demonstrating that PNE accelerates the development of sleep/wake systems. This PNE effect on the maturation of sleep/wake behavior was only significant in male offspring at P13. In

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150 addition, PNE males demonstrated a significant reduction in HR during sl eep selectively at P13. This significant bradycardia may be related to an accelerated integration of the sleep/wake system with cardiovascular control mechanisms (HR SD1/SD2) and could create a serious vulnerability if another HR lowering stimulus was encountered during sleep like a prolonged hypoxic exposure. Neither of these early postnatal changes w ere identified in fem ale PNE pups. Like in aim #1, PNE also ind uced sustained chang es in cardiorespiratory control at postnatal days later than originally hyp othesized. Unlike aim #1, these changes were more subtle and both sexes demonstrated some index of dysfunction. However these observations reinforce current hypotheses th at prenatal insults may be related to the development of certain diseases later in lif e, such as diabetes or hypertension (13) Aim #3: A Potential Orexin Mechanism Aim #3 examined the effect of PNE on the hypothalamic o rexin neuron population. Since orexin has been shown to modulate sleep/wake systems and alter cardiorespiratory function, t his aim was conduct ed to determine if PNE had any potential effect s on the orexin system. We hypothesiz ed that PNE would significantl y alter orexin transcription and cellular morphology and these effects would be present at P26. The most striking finding of aim #3 was the identification of a significant reduction in orexin mRNA after PNE in both sexes. Interestingly this change in gene expression occurred in the absenc e of any changes in cellular morphology. Furthermore, all females demonstrated elevated levels of orexin mRNA and the reduction in orexin transcription after PNE in females still did not drop to the inherent levels observ ed in the CON males. These data suggest that PNE induces epigenetic changes in the orexin system. Interestingly, t hese animals did not demonstrate some of the typical

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151 characteristics (increased apneas and fragmented sleep patterns) of orexin KO mice, sugge sting that the impact of this loss after PNE in the orexin system, at least at P26, were relatively subtle. Additional work needs to be done to determine how PNE changes the entire orexin system including changes in the developmental time course of orexin producing neurons, how orexinergic projection to other nuclei are altered and whether there are changes in orexin receptor expression or function in select regions of the brain. Discussion Normal Sex Differences in Central Cardiorespiratory Integration Epidemiological evidence has long suggested that sex differences exist in overall neural development (25, 185) The results o f the present group of studies support this hypothesis that sex differences exist during normal neural development. In aim #1, independent of any consideration of sleep/wake state we described differences in parasympathetic drive at P13 in normal females c ompared to males. This difference was expressed by a reduction in high frequency power (HF), a potential index of overall parasympathetic activity that was present in females but not in males. This observation suggested that females may lag behind males in parasympathetic development. However when we considered state dependent behavior in aim #2 and used time domain analysis (Poincare plots), the results did not support the previously suggested sex -dependent lag in parasympathetic developmental. Instead we demonstrated that in females, the HR SD1/SD2 index of sympathovagal balance was skewed in favor of parasympathetic drive with overall greater state dependence compared to age matched males. The potential for an earlier maturation of state dependent changes in HR in the females may be the result of differences in the time course of orexin development.

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152 Although we did not actually measure orexin expression at P13, our results confirm previous reports that overall orexin levels are higher in adult females (105) Moreover, if females began the development of the orexin sy stem at an earlier time point th a n was evaluated in the present studies, it might be expected that the females would demonstrate an earlier maturation of state-dependent control of cardiorespiratory function. Furthermore, females would possibly have presented with less dramatic shifts in sleep/wake behavior compared to males at the time p oints evaluated because the orexin system was already on board. In contrast, if males began central developmental changes in orexin expression closer to P13, state dependent changes may have appeared more dramatic. Indeed female developmental patterns in sleep/wake times were more gradual throughout the postnatal period examined in the present study. Alternatively, males had more striking changes in sleep time, specifical ly REM. In addition when transitioning from P13 to P16, females did not demonstrate a noticeable change in resting HR from QW to NREM, while males increased HR by ~25 bpm during the transition from P13 to P16 Overall, our results sugg est that the normal i ntegration of sleep/wake influence on cardiovascular function involves a precise ontogenetic drive which is fundamentally different between the sexes, and may begin earlier in females. Additional studies however are needed to identify the specific time course of orexin neural development in males versus females to confirm this hypothesis. Interestingly, the index of RSA used in aim #1 showed no major differences between the sexes (P13 through P26). More specifically neither males nor females had any index of RSA at P13, but by P16 indices of RSA were apparent and at P26 RSA appeared to be adult like (approaching a slope of 1). We suggest that although females

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153 may develop mechanisms for sleep/wake integration with the cardiovascular system earlier (possibly orexin dependent), RSA can not be expressed until the cardiovascular system integrate s with the respiratory system. It appears that integration of the se two systems occurs around P16 in both males and females, thus we did not see any sex based differences in the time course of RSA. Previous investigators evaluating respiratory development, as an index of brainstem maturation, have demonstrated that brainstem development in the rodent can change on a day to day basis (134, 136) Hence, the results of the present groups of studies would have been more conclusiv e if we had evaluated our subjects at sho rt intervals ( 2 3 days) throughout the entire postnatal period to help identify some subtle sex based differences. Based on the outcome of our investigation of sex -based differences in cardiorespiratory/sleepwake pattern development, we propose a model that integrat es sex -based differences in orexin development with autonomic circuits involved in vagal or parasympathetic drive. The left column of Figure 51 illustrates this hypothesis including orexinergic modulation of brainstem circuits in CON males and females. Al though no work to date has described sex based differences in the development of the orexin system, our results from P26 suggest that females may begin to develop the orexin system earlier than males and generally produce more orexin protein. This is refl ected in Figure 5-1 by the thicker line from the orexin system to the NTS. Furthermore we propose that orexinergic inputs to the NTS increase excitation of GABAergic relay neurons that project to CVN. Thus, increases in orexinergic input triggers polysy naptic inhibitions of CVNs, resulting in an acceleration of HR through vagal withdraw This hypothesis is supported by in vitro work which has demonstrated

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154 that application of orexin increases GABA related neurotransmission onto CVNs. Moreover, this effec t appears to be pre-synaptic and may be related to the modification of GABA ergic input to the CVN s from the NTS (42) In addition, during both electrical stimulation and photoactivation of the NTS neurons, orexin application in the slice has been shown to enhance inhibitory currents within the CVN (39) As a consequence, an elevated level of orexin in females would be reflected by an overall increase in HR compared to males both at rest and during hypoxia, a predicted response which generally fits our data. In the present study CON females demonstrated significantly elevated orexin levels and a sustained HR response during acute hypoxia at P26. At the same developmental time point, CON males demonstrated a biphasic response which was marked by an initial increase in HR and subsequent deceleration. This behavior confirms previous findings which demonstrate that adult rodents increase HR in response to hypoxia (145) Since the tachycardia seen during hypoxia has been suggested to involve the activation of the defense and arousal brain regions (145) it seems possible that orexin may be involved in the recruitment or maintenance of this initial tachycardic producing pathway. According to our model (Fig 5 1), this tachycardia would be mediated in part via parasympathetic withdrawal. Indeed both peripheral chemoreceptor inputs and r espiratory relat ed modulation of the cardiac vagal neurons (CVNs) are thought to oc cur through a GABAergic pathway (240) possibly located in the NTS (65) Therefore as illustrated in Figure 51, associated with the recruitment of the orexin system in response to hypoxia, females would present with a sustained tachycardia due to elevated levels of orexin release in the NTS and increased

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155 GABAergic inhibition of CVNs. If males have lower levels of orexin, the initial tachycardia during hypoxia might be lower than in females and the subsequent bradycar dia may reflect the recruitm ent of another circuit, which may also be recruited in the females but is not expressed due to an imbalance of orexin levels. It is interesting to note however, that during early postnatal development (P13 and P16) females demonstrated a biphasic HR response to acute hypoxia, while males at P13 had a sustained HR response, but did demonstrate two phases at P16. This biphasic HR response to hypoxia has been previously identified in both early rodent and piglet development (53, 81) Unfortunately, the use of different species and hypoxic protocols makes it difficult to compare the overall postnatal development. However, we suggest that orexin development may play a putative role in generation of not just the tachycardia responses described above, but also the developmental changes in the bradycardic ph ase or the ability to expr ess the bradycardic component. It should be noted that figure 5-1 does not depict potential development of orexin neurons or how they are related to the developmental changes of the hypoxic response. Although ultimately the mechanism for these sex dependent changes in brain maturation is unknown, one possibility may be related to f luctuations in hormones that occur during early postnatal life. Male neonate rodents, and humans alike, experience a large surge of testosterone during the immediate postnatal period (63, 99, 128) It had been hypothesized that this surge is responsible for the mascularization of the brain (78, 101) It may be suggested that this mascularization is responsible for a permanent alteration in ontogenetic timing mechanisms which direct postnatal development. Therefore, regardless of current hormone profiles, males and females will develop

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156 integrati ve processes differently. More work m ust be done to determine 1) each sexs developmental timing of brain processes, including those regulating the autonomic nervous system and orexin and 2) if these sex differences are dependent on a postnatal testosteron e surge Effect of PNE on C ardiorespiratory I ntegration The three main findings of the present research support our original hypothesis that PNE animals would demonstrate an accelerated integration of three main physiological functions, sleep/wake, respi ratory and cardiovascular control First, we found that all PNE pup s demonstrated a high er index of RSA compared to CON pups at selected developmental time points Second, male PNE pups at P13 had a significantly blunted HR during hypoxia compared to CON m ale pups Third, PNE males demonstrated a larger drop in HR during sleep than C ON counterparts exclusively at P13. We suggest that t he accelerated integration of these central processes (both state and respiratory) on cardiovascular function may actually r eflect a premature or over development of this integration process, possibly indicative of a period of ca rdiovascular instability. During normal rodent postnatal brainstem development regions like the preBtC and NTS demonstrate a significant reorganiz ation of GABA signaling (135, 136) Furthermore, PNE has been demonstrated to alter GABAergic transmission o n to the CVN (171) during both normox i a and hypoxia (98, 170) Thus, if GABAergic input to CVNs undergoes a similar postnatal development as in other brainstem regions, then PNE may alter the timing of these postnatal changes. As illustrated in Fig 51 we hypothesize that changes in the CVNs GABA system may be related to a change in orexin signaling. The results of aim #3 demonstrate that although cellular morphology

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157 did not change, orexin transcription wa s indeed significantly altered after PNE. However, orexin mRNA was reduced at P26 and not elevated as would be hypothesized based on our current putative role of orexin. As illustrated in figure 5-1, we suggest that the identified impact of PNE to induce a reduction in orexin mRNA at P26 may be associ ated with a significant increase in inherent orexin receptor sensitivity. R ecent studies with chronic nicotine exposure suggest that nicotine can paradoxically alter orexin signaling function by changing prepro orexin mRNA expression in one direction and r eceptor sensitivity in the other (109, 110) Additionally, we suggest that PNE ac celerates the development of orexin. This would account for the acceleration in the development of sleep/wake time, sleep/wake integration into cardiovascular control, and cardiorespirato ry maturation. In females this was expressed as an earlier maturation of RSA at P13 and in males the over maturation of RSA occurred at P16. It is interesting to note that indicators of premature maturation of integrating circuits (RSA) were coupled to indicators of an overall increase in parasympathetic tone (HR SD1 and/or HR SD1/SD2) in PNE males at P13 and P16 and in the PNE females at P13 compared to their sex matched controls. However, in the males at least at P13 and P16, resting HR (aim #1) was si gnificantly higher than CONs. Thus, it is also possible that PNEs acceleration of relative parasympathetic function may not be the sole dysfunction, but rather a compensatory mechanism for another underlying problem. The present data from both aim #1 and #2 suggest that some of the abnormal cardiovascular function seen after PNE may be cause d by i ncreased intrinsic HR. The generation of pacemaker activity, and subsequent HR, involves a complex interaction between spontaneously depolarizing cells within th e sino atrial node and cardiac fibers

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158 which carry that impulse. These cardiac cells use a number of different ion currents to create these depolarization events which will ultimately lead to cardiac muscle contraction. In aim #3, we suggested that PNE may be causing important epigenetic changes at least within the hypothalamic orexin system. It is possible that P NE also induces epigenetic changes in cardiac tissue as well. A precident for epigenetic alterations in intrinsic HR was recently identified in di abetic rodents. In this instance the induction of diabetes was shown to decrease mRNA expression of critical cardiac gap junction proteins, which correlated with slower intrinsic HR (94) It seems entirely possible that the changes in autonomic function seen after PNE are actually compensatory changes to the alterations of intrinsic HR function. Furthermore it is possible that the effect of PNE on HR at least state dependent changes in HR re flects a change in circadian clock gene expression. For example, cardiac pacemaker cells have been shown to express circadian clock genes and associated circadian rhythm ic activity (119) These genes and the circadian patterns also demonstrate specific postnatal developmental patterns (191) Although circadian rhythm do es not imply sleep related ch anges per se, it does suggest that some sex dependent differences and PNE -induced effects on state-dependent changes in HR and the maturation of these patterns may reflect differences in intrinsic HRs Effect of PNE on Sex Bas ed Neural Plasticity Since it appears that PNE results in abnormal brain maturation, it is not surprising that sex differences may exist in the impact of PNE on neural development. The more gradual development in normal females of sleep/wake and respirator y influence over immature cardiovascular control networks may create a neural environment where deficiencies caused by PNE are less striking and more easily corrected for. For

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159 example, in the present studies females did not demonstrate the significantly bl unted HR response to hypoxia or significantly lower HRs during sleep compared to QW two effects that were exhibited in PNE males Interestingly, PNE females showed an over development of RSA at P13 which was sooner than their male counterparts This devel opment was remedied in females by P1 6 (when males first demonstrated it).These changes were paralleled with an increased parasympathetic index (HR SD1) across sleep/wake states in PNE female s at P13. We have suggested that at P13, females are able to compensate for PNE induced alterations in cardiovascular function by recruiting the parasympathetic system, whereas males may only be able to mediate changes through the sympathetic system. T h ese observations raise the possibility that females who are compromis ed by PNE either 1) transition out of the SIDS c ritical developmental window sooner and/or 2) develop compensatory mechanisms more effectively than their male counterparts A greater inherent plasticity in females during early postnatal development w ould c ontribute to their lower rates of SIDS S ex based plasticity has been reported following other perinatal insults. For example, an adult female who was exposed to postnatal hypoxia shows no change in their response to a subsequent ex posure to hypoxia, but t he responses of adult males were markedly blunted (14) Moreover, because the greater developmental plasticity in females in the present study occurred prior to the onset of puberty (47) it is likely that the potential for greater plasticity in females is not based exclusively on estrogen PNE Increases Risk of Cardiovascular Disease One unexpected result of the present st udies was the persistence of cardiovascular abnormalities at P26 Most surprising was the paradoxical effect of PNE on the HR response to hypoxia seen at this time and the slightly aberrant RSA observed

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160 in later development in males alone Howev er, in hindsight, a persistent effect of PNE might have been predicted. For example, PNE has been shown to cause cholinergic deficiencies within the adolescent brain (217) Epidemiological data suggests that as infants exposed to maternal smoking get older, they have a greater tendency to develop both hyperactivity and attention deficient disorders (43, 55) reduced memory and spatial abi lity (35) and increased incidences of cigarette usage (174) Some of these longer lasting effects of PNE are also differentially expressed in males and females (54, 174) Although, the majority of previous reports have been on the cognitive function after PNE a few studies have examined the impact of PNE on the development of overall cardiovascular health and development of function. Indeed, PNE associated reductions in cardiac adrenergic sensitivity that occurs in rodents has been shown to persist into adulthood (27) Adult PNE males also demonstrate an angiotensin II dependent hyper -responsiveness that is not present in PNE females (247) Thus, in dicators of elevated sympathetic drive identified in the present study may be mediated by chronic changes in central sympathetic control elicited either as a compensatory response to changes in cardiac sensitivity or abnormalities in the brain angiotensin system (247) and/or the orexin system. Of p articular importance then is the potential for PNE to predispose at least male offspring to an elevated incidence of cardiovascular disease, such hypertension, in later life (13) and the origin of this predisposition may be multi -factorial. Future studies into the specifics of these chronic changes are warranted to identify selective therapeutic approaches to reverse this potential for chronic autonomic dysfunction.

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161 Study Limitations Surgical Stress The larg est consideration for aim #1 and #2 is the potential effect of surgery stress on the youngest pups. To allow for a more realistic evaluation of HR regulation in conscious unrestrained animals, we chose to use chronic instrumentation. Unfortunately, we found that pups instrumented before P12 had difficulties in recovering weight. Ther e is some evidence that PNE affects the responsiveness of the hypothalamic -pituitary adrenal axis (18 4, 247) Although both treatment groups received surgery on the same day, it may be possible that some of the alterations seen as a result of PNE were due to different stress responses to the surgery However, we tried to minimize all stress associated w ith the instrumentation by minimizing the maternal separation time, making sure that the pups were returned to their home cage and received maternal care. Moreover, any differences in stress responses to surgery may have been minimal since our results are similar to those previously reported (83, 219) Additionally, surgery recovery time has not been shown to a ffect developmental or sleep related changes in autonomic function (51) Time of Recording A second limitation of the present studies, particularly aim #2, is that we only examined a snap shot of rodent behavior. In normal rodent sleep development, different results have been found when onl y a few hours of sleep time were examined versus a full 24 hour protocol (23) Additionally, circadian rhythm function (and its development for that matter) can influence overall HR, RR, and their cent ral neural controlling centers (81, 160) However, using this extended protocol with chronic surgeries would req uire maternal separation. Since previous research has shown that

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162 maternal separation alone can influence sleep patterning, stress responses and overall cardiovascular funct ion (92, 235) we felt it important to maintain as much maternal care as possible d uring our study. It is possible that the present data does not represent the entire picture of PNEs effect on sleep -related integration of the cardiorespiratory system. Future Directions Autonomic Blocka d e HRV provides a noninvasive identification of postnatal changes in autonomic control of HR and a tool to potentially translate animal work to human patients. Although this method may one day al low physicians to better identif y infants at high risk for SIDS currently there is a need for determining direct causations and HRV does not allow for the direct manipulation of the autonomic nervous system. Nor does it allow for the identification of changes in intrinsic HR function. This may be important given that the data presented in aim #1 and #2 supported a potential change in not just autonomic function, but in intrinsic HR Therefore, future studies in animal models must employ the use of pharmacological agents to determine fundamental changes in these auton omic and pacemaker functions. RSA and GABA D evelopment in C ardiac Vagal N eurons The present studies and a number of others suggest that PNE causes over activation of respiratory related parasympathetic function. However, no study to date has examined the potential postnatal development of these changes. Future studies could examine this relationship in two ways First, an evaluation of changes in neurotransmitter expression following PNE, specifically GABA, receptor sensitivity and density in the NTS and preBtC is warranted. The second, utilizing in vitro brainstem

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163 slices one could evaluate how respiratory related GABA input to the CVNs changes throughout postnatal development (P 5 to P20) Using very specific developmental time point s for these studies w ould elucidate a mechanism for the enhancement of RSA after PN E. This point is particularly intriguing if females and males are following different developmental timelines in both normal development and in the resolution of PNE induced abnormalities. There fore, these studies should include a differentiation of sexes. Investigating Multiple Risk Factors SIDS is considered a multi -factorial syndrome (121) it is i mpor tant to consider that PNE is not the only risk factor Therefore repeating the present studies with the addition of other risks might help further elucidate the SIDS mechanism. A more realistic model of SIDS then might include two or more risk factors. For example, a study might use PNE as an in ut er o insult and then during hypo xia maintain a hyperthermic environment to determine if these factors are lethally additive (178) Orexin System Developmen t Finally, in the present group of studies we identified the need for more research to be done on the hypothalamic orexin system. Although we demonstrated a significant reduction in orexin mRNA production in aim #3, we only looked at one developmental time point. Thus investigations into a number of other components of the system are now warranted. I nitially, there must be an identification of whether this decrease in mRNA corr esponds to a decrease in actual orexin protein production. I t is possible that overall cellular orexin protein concentrati on is unchanged (197) or even elevated T his is plausible since we did not observe any overall difference in orexin staining in the brain. Additionally, studies need to examine the potential of PNE to affect orexin

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164 receptor mRNA and protein level. Receptor sensitivity should also be evaluated to determine if regardless of transcription/translation, orexin receptors are changing their over all function. I t is also important to include various brain regions that orexin neurons project to in future studies since signaling changes for orexin may be s ite specific Perhaps the most critical aspect in PNEs potential effect on orexin and its relationship to SIDS is how orexin change s occur as a function of developmental age. Conclusions Results of all three studies presented support previous work demonst rating that PNE accelerates the autonomic nervou s systems integration with other key centrally controlled functions, including sleep/ wake drive and respiratory function. More importantly, these studies support much of the work done to investigate the gender ratio of SIDS, by suggesting that male PNE pups are more vulnerable to PNE abnormalities. Although these physiological abnormalities are probably a result of a number of long lasting in utero alter ations of brain chemistry, we suggest that epigenetic ch anges in the hypothalamic orexin system and overall ontogenetic patterning may play a role in sex based difference s Moreover it appears the epigenetic changes induced in the central cardiovascular circuits following PNEs may persist and increase an indi viduals risk of disease later in life particularly for males

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165 Figure 51. Proposed mechanism for orexin mediated alteration of GABA neurotransmission to cardiac vagal neurons (CVN) during normal development (CON) and after prenatal nicotine exposure (PNE). Red depicts inhibitory actions. + indicates receptor sensitivity. Dashed line separates CON from PNE. NTS: nucleus of the solitary tract ; NA: nucleus ambiguus.

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187 BIOGRAPHICAL SKETCH Carie Renee Reynolds was born in Napa, CA. Wi th a l ack of the foresight of how much Carie would grow to love wine country, her parents relocated to St. Petersburg, FL. After graduating from the International Baccalaureate program at St. Petersburg Senior High, she spent the next four years attending the University of Florida in pursuit of a degree in z oology. In August of 200 4 she joined Dr. Linda F Haywards lab as a technician and quickly applied to the physiological sciences graduate program at the University Of Florida College Of Veterinary Medic ine s. In August of 2005, Carie officially began pursuing a grad uate education in the field of autonomic n europhysiology. During her graduate studies, Carie received a number of awards including the UF Alumni Fellowship and was honored for Excellence in t he Field of Neuro plasticity at the Annual Neuroplasticity Symposia. She received her Ph.D. in Veterinary Medical Sciences from the Department of Physiological Sciences in the College of Veterinary Medicine in December of 2009 and immediately began a postd octoral research position in the laboratory of Dr. David Mendelowitz with the Department of Physiology and Pharmacology in the College of Medicine at George Washington University in Washington D.C.